Abstract: The multispectral imaging is an effective tool for noninvasive observation of biological tissues, and it can further accurately obtain the multispectral data of pathological tissues by improving the irradiance uniformity of the light source. Therefore, the multispectral uniform light source system based on the integrating sphere was proposed to provide uniform irradiance for multispectral data acquisition of biological tissues. First, in order to determine the optimal parameters of the structural model, the design scheme of the integrating sphere was selected based on the Monte Carlo ray tracing method (MCRT), and the cavity size, LED layout, and the output slot size of the integrating sphere were optimized. Then, an example with 11 integrated narrow-band LEDs was constructed for optical performance verification based on the optimized parameters, and the experimental results showed that all the used bands could achieve 2% irradiance nonuniformity distribution in the major part of the output slot. And the broad-spectrum compatibility of the system was studied and the experimental results showed that the maximum root-mean-square error of the spectrum in the output slot was 3.74%, which had good broad-spectrum compatibility. Finally, based on the proposed system, venous visualization tests were performed and the endoscope compatibility of the system was extended with optical fiber and used to investigate the internal dental surface pathology of the oral cavity. The multifunctionality and uniformity of the system has potential applications in biomedical image acquisition.