A method for detecting zebrafish M3 receptor protein based on determination of p38MAPK protein expression comprises the following steps: (1), preparing a zebrafish brain tissue protein sample; (2), performing SDS (sodium dodecyl sulfate) gel electrophoresis; (3), performing film transferring; (4), closing; (5), performing primary and secondary antibody inoculation; (6), performing rendering reaction; (7), calculating M3 receptor variation results. The technical defect that traditional zebrafish M3 receptor detection is limited to zero specific antibody and accordingly is unable to use western immunoblotting is overcome, the use of p38MAPK phosphorylation level to determine changes in zebrafish M3 receptor protein content is provided for the first time, and the method is high in sensitivity, good in stability and free of complex equipment; meanwhile, the method is simple and quick to perform, high in specificity and low in cost; internal acting mechanism of a cholinergic system is studied in terms of molecules, and in connection with study on zebrafish behavior changes, finally water quality can be estimated and monitored online in real time.