Multi-spectral photoacoustic (MSPA) imaging can extract quantitative information of interesting component from morphological photoacoustic image of targets. A probe with an optimal optical absorption can be detected by MSPA imaging with higher sensitivity and specificity. Here we developed a Nano polymers dots (P-dots) for MSPA imaging that has a high photoacoustic conversion efficiency in a broad optical absorbing band (500 nm - 700 nm). In vivo MSPA imaging experiment was successfully performed in mouse model. The relative concentration map of P-dots was exactly obtained from the background of tissues, which demonstrated the potential use of P-dots in the bio-imaging field based on MSPA imaging.