The booming aquaculture industry has created a strong demand for fishmeal and increased environmental pressures. Spirulina, as a potential alternative to fishmeal, has been shown to have growth-promoting and animal health-enhancing properties. In this study, 600 large spiny loaches, divided into five experimental groups, F0, F1, F2, F3, and F4, were reared for 10 weeks using Spirulina platensis powder (SPP) as a substitute for 0%, 5%, 10%, 15%, and 20% of fishmeal, respectively. The results of intestinal physiological indexes showed that superoxide dismutase was lower than F0 in all treatment groups, and the activity of F3 was significantly lower than F0 (p < 0.05). The activity of malondialdehyde was significantly higher than that of F0 in all groups except F3 (p < 0.05). The addition of SPP also led to a decrease in the activity of acid phosphatase in the intestine, which was significantly lower in all treatment groups compared to the F0 group (p < 0.05). The results of serum physiology showed that the activity of superoxide dismutase in serum gradually increased with the increase in the percentage of SPP addition, and the F3 group produced a significant difference from the F0 group (p < 0.05). The transcriptomics results showed that DEGs in the low percentage substitution group (<15%) were mostly enriched in metabolism-related pathways, such as bile secretion; DEGs in the high percentage substitution group (>15%) were mostly enriched in inflammation-related pathways, such as complement p and coagulation cascades. Metabolomics confirmed that nicotinate and nicotinamide metabolism and glycerophospholipid metabolism were the two pathways that were significantly enriched in the treatment groups of fishmeal replacement by SPP. The present study demonstrated that a low percentage (<15%) of fishmeal replacement by SPP in feed mobilized MA digestive metabolism, whereas a high percentage (>15%) of replacement induced intestinal stress. Considering the health and farm efficiency aspects, the proportion of SPP in feed formulation for MA should be less than 15%.