According to the discrete three-dimensional (3D) fluorescence spectrum detection of organic matter in water, the high sensitivity and large dynamic range detection circuit and control software of weak fluorescence signal based on the variable gain and integral amplification technology are designed. Experiment verification is carried out and it’s applied to the on-line detection system for discrete 3D fluorescence of organic matter in water. In the designed circuit, according to the input signal intensity, automatic program control CMOS multi-channel analog switch is used to realize the adjustment of front gain amplifier. Based on the switching charge integrator, the synchronous integral amplification of weak pulse fluorescence signal is realized. Results show that the minimum detection current of the designed circuit is 0.09 μA, dynamic detection range is 0.09 μA～0.21 mA, the detection limit is increased by 79.25 times. In the application of discrete 3D fluorescence detection system of organic matter in water, the system detection sensitivity is guaranteed, and dynamic detection range of fluorescence signal is improved. The high precision measurement of organic matter in water is realized.