Aiming at the difficulty of the absolute quantum yield measurement of weak luminescence samples, a Four-Line method is proposed, mainly using the neutral density attenuator to realize the relative enhancement of the sample emission signal. The fluorescence spectrometer and integrating sphere measure the absolute quantum yield. To investigate the feasibility of the Four-Line method, the quantum yields of standard samples rhodamine 6G and quinine sulfate were measured. The results show that the relative errors are 1.3% and 1.1%, respectively, far less than 5.0%, indicating this method has high accuracy. The influence of experimental conditions on quantum yield measured by the Four-Line method is discussed. It is found that the best experimental condition is that the neutral density attenuator is placed at the excitation position, and the attenuated light source intensity reaches the upper limit of the linear response interval of the detector. They used rhodamine 6G solution with different concentrations to simulate weak, medium, strong, and strong luminescence samples. The quantum yields of the solutions were measured by the Two-Line method (conventional method) and the Four-Line method, respectively, and the results were compared with those reported in the literature. The research shows that the Four-Line method is more suitable for the absolute quantum yield measurement of weak and medium luminescence samples, and the relative error is reduced from 10% by the Two-Line method to 0.32%.