Prostate cancer is the second most common cause of death for men worldwide. The encapsulation of herbal extract in polymeric nanoparticles is a prominent therapeutic remedy and targeted drug delivery for cancer. The aim of the present study was to investigate the cytotoxicity and cell cycle arrest of human prostate cancer cells by using chitosan nanoparticles encapsulated Andrographis serpyllifolia extract. Aqueous extract of A. serpyllifolia (ALE) at various concentrations were encapsulated with 2% (w/v) chitosan nanoparticles (ALE CSNPs). Initially, entrapment efficiency of ALE by CSNPs and drug release percentage from CSNPs were determined and then ALE CSNPs were subjected to various spectroscopic characterization. The effect of ALE CSNPs on PC3 cells was examined by MTT assay and flow cytometry DNA analysis. Among, various concentrations of ALE CSNPs, 0.25 mg/mL concentration exhibited higher entrapment percentage (87.73%). In vitro drug release study showed a controlled and sustained crude drug release from ALE CSNPs (91.30%) within 5.5 hours. The addition of plant extract onto chitosan nanoparticles increases the size of the nanoparticles of ALE CSNPs i.e. 22.3 nm compared to control i.e. 29.3 nm which is confirmed by XRD. SEM image revealed the synthesized ALE CSNPs had huge surface area. Compared to CSNPs, ALE CSNPs showed decreased cell viability and increased percentage of obstructed cells at G0/G1 cell cycle phase effectively. In conclusion, ALE CSNPs act as a promising drug delivery system using chitosan nanocarrier for targeted release of A. serpyllifolia leaf aqueous extract to PC3 cell lines.