Polymer-based nanomedicine is a large and fast growing field that has gained plenty of research attention during recent decades. In the present study, new amphiphilic polymers were designed and synthesized by chemical modification of poly(ethylene glycol) (PEG) conjugated with sucrose and a cholic acid moiety (abbreviated as Suc–PEG–Chol). Two series of polymers with different PEG chain lengths were synthesized and their structures were confirmed by 1H-NMR, 13C-NMR and MALDI-TOF analysis. The fluorescence spectroscopy data of these conjugates showed that they are able to self-assemble in water and the critical association concentration (CAC) value was found to be in the range of 0.06–0.13 g L−1. Owing to their amphiphilic characteristics in aqueous solution, polymeric nanoparticles (PNPs) of Suc–PEG–Chol polymers were prepared by a nanoprecipitation method without any surfactants. The particle size distribution was determined by dynamic light scattering (DLS) and the result was 117 nm for the Suc–PEG2000–Chol conjugate and 96 nm for the PEG4000 analog, both with relatively narrow particle size distribution. All of the obtained PNPs showed a negative surface charge and no size dependence on the polymer concentration forming stable nanoparticle suspensions. From the atomic force microscopy (AFM) and scanning electron microscopy (SEM) observations, the PNPs were spherically shaped with a relatively smooth surface. Our results suggest that these PEGylated nanoparticles formulated with cholic acid and sucrose as biocompatible building blocks can be considered a potential candidate for biomedical applications.