The invention discloses a detection method for a time frequency two-dimensional phase-sensitive sum frequency spectrum interface based on pi-phase modulation. The detection method comprises the following steps: adding a delay wave plate and a phase modulator in an infrared light pulse optical circuit and carrying out delay processing and pi phase modulation on visible light pulse; matching the visible light pulse with the infrared light pulse in space and time, irradiating on a sample interface by the visible light pulse and the infrared light pulse at the same time and the same place, and acquiring an excited a two-dimensional sum frequency spectrogram by using a CCD (Charge Coupled Device) type spectrograph; carrying out data processing on the two-dimensional sum frequency spectrogram, and obtaining an amplitude, frequency and phase information of a molecular response function of the sample interface by using an inverse algorithm. According to the detection method disclosed by the invention, the amplitude, frequency and phase information of an interface molecular response function can be accurately obtained at the same time; the detection method is a high-efficiency accurate acquiring method for interface molecular information.