The objective of this work is the nanoencapsulation of quercetin in chitosan (CHI) nanoparticles (NPs) cross-linked by ionic gelation with sodium tripolyphosphate (TPP), in order to study the release profile of quercetin and evaluate its in vitro cytotoxic and antioxidant activity using tumor cells and the DPPH method, respectively. The NPs synthesized by ionic gelation method showed a low polydispersity index (PDI) of 0.208 ± 0.03, hydrodynamic diameter of 103.2 ± 5.7 nm and positive zeta potential with high module value (+30.4 ± 1.6 mV). These characteristics were directly influenced by the variation of the TPP volume. The NPs showed good colloidal stability during the period of 90 days at temperature of 37 °C. Samples were also characterized by DLS, BET, FTIR, TEM and TGA. The encapsulation efficiency of the quercetin in CHI/TPP NPs was 83.8 ± 0.33%, and its release was gradual and faster in solutions of NaH2PO4 at pH 7.4 by non-Fickian mechanism. From the evaluation of antioxidant activity in vitro using the DPPH method, it was observed that CHI/TPP/Quercetin NPs showed greater antioxidant effect when compared to free quercetin. Using the 3,4,5-dimethylthiazol-2,5 biphenyl tetrazolium bromide (MTT) assay, we observed that quercetin loaded in NPs showed a significant cytotoxic effect against human breast tumor (MCF-7) and human lung tumor (A549) in the 72-h period.