The invention relates to a data acquisition method of a non-collinear optical parametric amplification fluorescence spectrophotometer. In a non-collinear optical parametric amplification process, on a basis that a pump light, a fluorescence path and an idler beam path meet conservation of energy and conservation of momentum, the intensity of the fluorescence path and the intensity of the idler beam path have high correlation; a to-be-detected fluorescence signal and a to-be-detected idler beam signal after energy is amplified are respectively acquired, and a correlation operation is performed on sampling results of the fluorescence path and the idler beam path, so as to obtain a cross-correlation result of a to-be-detected fluorescence and a to-be-detected idler beam when in zero delay; a positive square root of the cross-correlation result represents a signal value of the to-be-detected fluorescence on a kinetic curve when in different time delays. According to the data acquisition method, the noise of a non-collinear optical parametric amplification fluorescence spectrophotometer system can be more effectively eliminated, the SNR (signal to noise ratio) of the obtained kinetic curve of the fluorescence is improved, the fluorescence spectrophotometer with higher time resolution is realized, the measurement time is shortened, and the accuracy of a measurement result of an active sample is ensured.