Mystus macropterus, which belongs to the Bagrdae family of the Siluriformes order of Osteichthyes, was a fast-growing and large economic fish species found naturally in the mainstream and tributaries of the Yangtze and Pearl Rivers. It had high levels of protein, fat, essential amino acids, and umami amino acids, making it highly valued for consumption. However, the lack of breakthroughs in large-scale breeding of this species hindered its industrial development. This study aimed to improve the breeding efficiency of M. macropterus by optimizing its artificial insemination process. The study investigated the effect of different sperm preservation solutions (A-F), sperm-to-egg ratios (5.0 × 10⁴, 1.0 × 10⁵, 1.5 × 10⁵ and 2.0 × 10⁵), activation solution (mL) to egg (g) ratio (2∶1, 1∶1 and 1∶2), duration of gamete contact (2, 3 and 4 min), and egg quality (normal or overripe) on fertilization rate of M. macropterus. The experimental results showed that different preservation solutions significantly affected sperm vitality, fertilization rate and hatching rate. Among them, the C solution had the best effect with a fertilization rate of 60.22% and a hatching rate of 41.55%. Meanwhile, the fertilization rate significantly increased with the increase of sperm-to-egg ratio from 5.0×10⁴ to 1.0×10⁵. There was no significant difference in fertilization rate and hatching rate when the sperm-to-egg ratio was between 1.0×10⁵ and 2.0×10⁵, and a regression equation was established to determine the relationship between sperm density and fertilization rate. In addition, the highest fertilization rate and hatching rate was achieved when the ratio of activating solution to egg weight was 2∶1, which was significantly higher than that of 1∶1 group and 1∶2 group. There was no significant difference between the latter two. The fertilization rate ranged from 54.21% to 59.29%, and the hatching rate ranged from 37.40% to 40.91% in the three designed sperm-egg contact durations, and there was no significant difference among them. The maturation status of eggs had a significant impact on the fertilization rate of M. macropterus, with the fertilization rate of over-matured eggs (24.00%) significantly lower than that of regular eggs (34.38%). The study screened appropriate sperm preservation solutions, sperm-to-egg ratios, activating solution amounts, and duration of gamete contact for M. macropterus, providing references for large-scale and efficient breeding of this species.