The formation of nanoparticles (NPs) and surface properties such as size and charge are affected by the amphiphilic property of polymer, which is vital for evaluating their function. Here, we synthesized cholesteryl-modified aminated pullulan polymers (CHPNs) with different amounts of cholesterol succinate (CHS). We characterized the three hydrophobically modified polymers (CHPN1, CHPN2, and CHPN3) (CHS: Pu‐NH2=1/5,2/5,3/5) by Fourier transform infrared spectrometry. Dynamic light scattering (DLS) was used to measure particle size and zeta potential of CHPN NPs. The particle sizes of the three NPs CHPN1, CHPN2, and CHPN3 were 178.0, 144.4, and 97.8 nm, respectively. The particle size was related to the cholesteryl substitution of polymers to a certain extent: the stronger the hydrophobicity, the smaller the particle size. In 48 h, the drug release for CHPN3 and CHPN1 NPs was 57.8% and 72.7%. Thus, the NPs showed good sustained drug release: the greater the degree of hydrophobic substitution, the better the sustained release. The cytotoxicity findings were reversed: CHPN1 NPs, with low hydrophobic substitution, showed the best inhibition of Lewis lung cancer cells.