Mystus macropterus has delicious taste, rich nutrients and fast growth rate. It is a high-quality breed requiring further development. To investigate the biological characteristics of M. macropterus sperm and its adaptability to the external environment, the pH, osmotic pressure, sperm density, and sperm motility parameters of semen were determined, and the structure of the sperm was observed. The effects of environmental factors, such as pH, glucose, and ions (including NaCl, KCl, and CaCl₂), on sperm motility were also investigated. Based on the experimental data, the sperm of M. macropterus possessed a head, neck, and tail, and lacked acrosomes and had lateral fins. The mean sperm density of M. macropterus was 2.50×10⁹ , the pH of semen was stable between 7.0 and 7.2, and the osmotic pressure was stable at (634.16±6.66) kPa. Na⁺ content was the highest in the seminal plasma, followed by K⁺ , Mg²⁺, Ca²⁺, Fe³⁺, and Zn²⁺ and no Cu²⁺ was detected. The total amount of hydrolyzed amino acids in seminal plasma was approximately 169239.21 μmol/L. The largest proportion was composed of leucine, whereas the methionine content was relatively low. When sperm was activated by deionized water, the percentage of motile sperm, fast movement time, and life time were (48.61±14.85)%, (34.00±4.00) s, and (396.50±9.50) s, respectively. The straight line velocity, curvilinear velocity, and average path velocity were (11.50±6.26), (24.21± 8.68), and (14.00±5.99) μm/s, respectively. Sperm activity was most powerful in a slightly alkaline water environment with a pH of 7.0 to 9.0, with the optimal pH being 8.0. Simultaneously, when the concentrations of NaCl, KCl, and CaCl₂ were 1, 5, and 6 g/L, respectively, the percentage of motile sperm reached a maximum. Sperm motility was the greatest when glucose levels were maintained at 7 g/L. In conclusion, the sperm of M. macropterus had the best living environment in a pH range of 7.0 to 9.0. At the same time, appropriate concentrations of NaCl, KCl, CaCl₂, and glucose could also effectively improve the sperm motility of M. macropterus. In this study, we discuss the biological characteristics of spermatozoa and changes in their motility under the influence of different environmental factors. However, further research is required to establish a more effective breeding strategy and improve the artificial breeding efficiency of M. macropterus.