The invention provides a transgenic crop risk assessment method based on mesofauna multi-generation observation. According to the method, mesofauna is raised in a culture medium with transgenic crop litter, survival rates, reproductive rates and body lengths of different generations of the mesofauna are counted, functional enzyme of the mesofauna is detected, and the influence of transgenic crops on continuous multi-generation mesofauna population fitness is comprehensively assessed. By improving a mesofauna culture medium formula, the culture medium is more adaptable to multi-generation continuous culture of the mesofauna by the transgenic crops. Besides, by monitoring response of acetylcholine esterase, superoxide dismutase, catalase and the like in the functional enzyme of the mesofauna to the transgenic crop litter, response of the mesofauna to the transgenic crops is comprehensively reflected according to parameters such as death rates and the survival rates of the mesofauna.