In this study, CuS nanoparticles with optical absorption covering both nearinfrared I (NIR-I) and NIR-II biological windows were prepared and served as the contrast agents for multispectral photoacoustic imaging. The physiological parameters including concentrations of deoxyhemoglobin and oxyhemoglobin as well as the water content in the tumor location were quantified based on the multispectral photoacoustic reconstruction method. More importantly, the concentration of CuS nanoparticles/drugs accumulated in the tumor was also recovered after intravenously injection, which are essential for image-guided cancer theranostics. In addition, phantom and in vivo experimental tests were performed to inspect and compare the imaging depth and signal-to-noise ratio (SNR) between the two NIR biological windows. Interestingly, we discovered that a higher SNR was obtained in the NIR-II window than that in the NIR-I window. Meanwhile, the multispectral imaging results also demonstrated that the imaging contrast and penetration depth in the NIR-II window were also significantly improved as compared to those from the NIR-I window.