To study the effect of quercetin (QUR) on modulating immune effects, enhancing anti-tumor activity, and reducing drug related side effects, three QUR nanosuspensions (QUR-NPs) with different particle sizes were prepared by a microprecipitation-high pressure homogenization method using mPEG-DCA as a stabilizer. Dynamic light scattering was used to analyze the particle sizes of the three QUR-NPs. The results of stability tests showed that the three QUR-NPs had good storage and plasma stability. It was confirmed that plasma protein adsorption occurred for all three QUR-NPs. The results of DSC, DTG, XRPD, and Raman spectroscopy showed that there was no significant change in the crystal form of QUR for any of the three QUR-NPs compared with the commercial QUR. The in vitro dissolution rate of the three QUR-NPs was significantly faster than that of the micronized QUR, with the dissolution rate increasing as particle size decreased. All three QUR-NPs showed stronger in vitro inhibitory activity on MCF-7 cells than the pure QUR solution, with the largest NPs having the strongest inhibitory effect. The pharmacokinetic parameters in rats showed that the MRT and t1/2 of the QUR-NPs increased as particle size increased. QUR-NPs and the pure QUR solution showed obvious anti-tumor effects against murine hepatic carcinoma H22 model in vivo, although they were not as effective as cyclophosphamide (CTX). However, the anti-tumor effect of the large QUR-NPs combined with CTX was the strongest among all the tested groups. From the results of the thymus and spleen index, it was found that the QUR-NPs could not only regulate the immunity of tumor-bearing mice, but also alleviate the immunosuppression caused by CTX and protect normal tissues, all while enhancing the anti-tumor effect. The immunomodulatory effect of the QUR-NPs on tumor-bearing mice was significantly better than that of the pure QUR solution. Therefore, nanosuspensions can be used as a new drug delivery system for QUR to assist tumor therapy and regulate immunity.