Fabrication of near-infrared (NIR) fluorescent nanoprobes (NPs) has drawn great attention due to their potential as highly sensitive optical probes for in vivo tumor molecular imaging. Herein, a facile strategy has been reported for the preparation of ultrasmall (<20 nm) indocyanine green (ICG)-containing NIR fluorescent NPs using self-assembly chemistry between folate (FA) modified water-soluble chitosan (WCS) and ICG. The ICG-containing NPs exhibit characteristics of crystalline solid, good photostability, low cytotoxicity and high tumor targeting ability for in vitro cell imaging. We also explored this system for in vivo tumor molecular imaging. Significant tumor accumulation was observed for both in vitro and in vivo tumor imaging in HeLa tumor cell imaging and xenograft-bearing mice model (2 μmol kg-1 at 48 and 72 h time points). The availability of these self-assembled NIR NPs provides a convenient tool for tumor imaging and detection.