Protein phosphorylation modification can affect muscle quality. In order to investigate the effect of exogenous enzyme addition on the protein phosphorylation level of tilapia myofibrillar protein, tilapia myofibrillar protein solution was incubated in vitro by adding protein kinase A (PKA) and alkaline phosphatase (AP), respectively, and changes in phosphorylation level were determined by dodecyl sodium sulfate-polyacrylamide gel electrophoresis and fluorescence staining at different time intervals. In addition, the effect of adenosine triphosphate (ATP) on tilapia muscle was investigated by immersing the muscle in different concentrations of ATP solution to determine the level of myofibrillar fibrillar protein phosphorylation. The results showed that from 0 to 72 h, the phosphorylation level of PKA group was significantly higher than that of control group and AP group, and the overall phosphorylation level of PKA group increased from 0.35±0.01 at 0 h to 0.37±0.01 at 12 h, and then decreased to 0.29±0.01 at 72 h, with a general trend of increasing and then decreasing. In addition, the phosphorylation level of myosin heavy chain and actin phosphorylation level decreased from 0.73±0.01 and 0.86±0.01 at 0 h to 0.58±0.02 and 0.68±0.01 at 72 h, respectively (P<0.05). Phosphorylation levels were significantly different in all three groups when incubation time was 0, 4, 24 and 48 h. After addition of 0.3 mol/L exogenous ATP, the results showed that the overall phosphorylation level of myofibrillar proteins (0.46±0.001) was significantly higher than that of the control group (0.42±0.01). PKA promotes phosphorylation modification of myofibrillar proteins in tilapia, while alkaline phosphatase dephosphorylates them. It was shown that ATP content, PKA and AP activity levels in the muscle of slaughtered tilapia were key factors affecting the level of protein phosphorylation. This study may provide a theoretical basis for exploring the mechanism of quality change and regulatory strategies in tilapia.