Natural polysaccharides with multiple biological activities from traditional Chinese medicine (TCM) can be self-assembled. Natural nanoparticles have been found to exist in TCM decoctions. However, the influence of polysaccharides in TCM decoctions on the formation mechanism, drug release and gastrointestinal stability of nanoparticles remains unclear. Therefore, using Zhimu and Huangbai herb pair decoction (ZBD) as an example, this study aimed to reveal the effects of polysaccharides on the formation, gastrointestinal stability and in vitro release of nanoparticles (N-ZBD) in ZBD. First, N-ZBD was successfully isolated by high-speed centrifugation with an average particle size of 225.9 nm, PDI of 0.53 and zeta potential of -13.00 mV, and N-ZBD is mainly composed of small active compounds and polysaccharides. Interestingly, gastrointestinal stability results showed that N-ZBD had good stability while and the nanoparticles after polysaccharide removal (RPN-ZBD) were degraded within 4 h, indicating that polysaccharide can protect the stability of nanoparticles in the gastrointestinal environment. Moreover, in vitro drug release results showed that compared with free drugs and RPN-ZBD, N-ZBD has obvious slow-release behavior in simulated gastric fluid and simulated intestinal fluid, suggesting that polysaccharides contribute to the slow release of N-ZBD. The critical micelle concentration (CMC) of polysaccharides was 0.63 mg/mL, which could self-assemble with small active compounds to form N-ZBD. In conclusion, polysaccharides of ZBD were essential for N-ZBD formation, and could protect the stability of N-ZBD in the gastrointestinal environment and contribute to its slow drug release.