A high sensitive single photon counting system with a new photomultiplier tube as a sensor was developed to detect the ultra-weak biophoton emission from wheat, which fulfilled the absolute quantity detection of photon emitted from wheat in the storage condition. An illuminating system for the delayed bioluminescence was designed with a proper LED array. The relation of the illuminatingintensity and the intensity of photons was established through calculation of equivalent single photon energy and lumen efficiency for white LED, and the quantity comparison of illumination intensity and delayed bioluminescence intensity was realized. There existed different illuminating saturation time at different illumination. The accurate calibration of time for testing data of the delayed bioluminescence was fulfilled by accurate control of the illuminating time and data acquisition time, and also setting the delayed time parameter in fitting function. The ultra-weak delayed bioluminescence from wheat was detected and analyzed systematically, and the results show that the intensity curves of delayed luminescence from wheat follows a hyperbolic attenuation trend, and the duration of the luminescence lasts for several hours. Subsequently, the hyperbolic function fitting of testing data was carried out and the changing regularity of fitting parameters was discussed.