A new determination method of cellulase activity—gel diffusion-tissue blot was described. This paper provided the basic principle, operation procedures and calculation methods for cellulase assay, and applied this method to analyze the activity of cellulase in the abscission layer in longan pedicle during fruit abscission. The results showed that this method had advantages such as less material requirement, high sensitivity, good reproducibility, simple operation, and saving time and labor, and it is very suitable for the determination of cellulase activity in abscission layer.