The invention relates to the field of activity detection of biological medicine, in particular to a biological activity determination method for an anti-PD-L1 monoclonal antibody. According to the method, a PD1/PD-L1 interruption detection system is used, binding of PD1 and PD-L1 protein is interrupted when the anti-PD-L1 monoclonal antibody is bound with PD-L1 expressed by target cells, a transcription factor NFAT is activated through signal transduction, expression of luciferase is started, the expression quantity of luciferase is positively correlated to the biological activity of the anti-PD-L1 monoclonal antibody, the chemiluminescence value of the anti-PD-L1 monoclonal antibody is determined after a cell lysis solution and a luciferase substrate are added, a dose-effect curve is drawn, and accordingly the biological activity of the anti-PD-L1 monoclonal antibody is determined. The method is easy to implement, strong in specificity and high in durability and has great significance on quality control and clinical application of the anti-PD-L1 monoclonal antibody.