Hybrid sturgeon is regarded as a critical aquatic economic species, whose high/low fertility (spawning yield) is associated with the economic development of the caviar industry. However, in the aquaculture production process, female hybrid sturgeon have a great differential spawning yield in the sexual maturity period. Meanwhile, there are a series of technical barriers to early breeding in hybrid sturgeon populations with high egg yields. Therefore, it is essential to explore the potential metabolism pathways that affect the fertility of hybrid sturgeon and filter the biomarkers for the high spawning yield phenotype in the female hybrid sturgeon populations. This can benefit the understanding of the biological mechanisms of fertility differences in hybrid sturgeon and accelerate the sustainable development of the sturgeon breeding industry in China. However, relatively few reports have clarified the expression pattern of ovarian metabolic profile between the sturgeon populations with high/low fertility. In the sexual maturity period, 90 hybrid sturgeon individuals (Huso dauricus♀ × Acipenser schrenckii♂) had their egg weight measured using a minimally invasive laparotomy method. Based on the real egg weight of each hybrid sturgeon, the present study defined the high/low fertility standard as follows: the ratio of egg weight/body weight (θ) less than 0.15 was classified into the low fertility group while θ more than 0.20 was classified into the high fertility group. In the formal experiment, a total of sixteen hybrid sturgeon with high (eight) and low (eight) fertility phenotypes were selected from the above population. Subsequently, fresh ovary tissues were collected to perform untargeted metabonomics analysis after recording the body weight and egg weight of each hybrid sturgeon. Bioinformatics methods and public metabolite database were applied to identify the metabolite category and corresponding relative abundances. The dominant metabolite categories in the hybrid sturgeon ovaries were lipids and lipid-like molecules, organic acids and derivatives, and organoheterocyclic compounds. Based on the threshold value levels reported in the previous studies, a total of eleven differential metabolites were identified between high (low) fertility groups. According to the blast results provided by the public metabolite deposition database, these eleven differentially expressed metabolites belonged to five categories including organoheterocyclic compounds, organic nitrogen compounds, nucleosides, nucleotides, and analogs, lipids and lipid-like molecules, and organic acids and derivatives. Compared to the low fertility group, a series of metabolites such as (–)-riboflavin, riboflavin, spermidine, adenosine, and lysoPI 22∶6 were down-regulated in the ovary tissues of the high fertility group. Additionally, the receiver operating characteristic curve also determined that (–)-riboflavin, riboflavin, cyclohexylethyl acetate, and L-alanyl-L-norleucine could be regarded as candidate biomarkers for the high fertility phenotype of female hybrid sturgeon. Subsequently, Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis was performed based on the differentially expressed metabolites. Our results showed that amino acid metabolism-related pathways (arginine and proline metabolism and beta-alanine metabolism) and vitamin metabolism-related pathways (vitamin digestion and absorption and riboflavin metabolism) were significantly enriched and the P values of these four pathways above were 0.0019, 0.0247, 0.0001, 0.0004, respectively. Meanwhile, it was found that adenosine is involved in fourteen metabolism pathways in the ovary tissues of hybrid sturgeon, inferring that it may have critical regulatory effects on the fate decision process of fertility phenotype of female hybrid sturgeon. The present investigation has preliminarily clarified the molecular signaling that affects the spawning yield trait of hybrid sturgeon and filtered the candidate biomarker for high fertility phenotype. This could provide a theoretical basis for breeding the hybrid sturgeon population with a high spawning yield. Taken together, our investigation not only characterized the metabolite profiling difference between the hybrid sturgeon populations with high (low) fertility phenotypes but can also shed light on directions for further research on the reproductive physiology of hybrid sturgeon and biomarker-assisted selection breeding for freshwater economic fish.