The present study was focused on developing nanoparticles (NPs) loaded with dual drug conjugate (DDC) for the enhanced anti-tumor activity and reduced resistance against renal cancer cells. Gemcitabine (GT), a nucleoside analog, and 5-Fluorouracil (5FU), an anti-metabolite of thymidylate synthase inhibitor, were conjugated using succinyl chloride (SC). The conjugate GT–SC–5FU was confirmed by 1H NMR and mass spectroscopy (MS). DDC was then loaded onto bovine serum albumin NPs and the formulated NPs were characterized for surface morphology, particle size, zeta potential and PDI. In-vitro drug release and cytotoxicity studies were also performed. Furthermore, the apoptosis study was conducted in zebrafish embryos. Our results demonstrated that DDC was successfully synthesized and the structural conformation of the conjugate (GT–SC–5FU) was confirmed by 1H NMR and MS. DDC-loaded NPs showed smooth-surfaced spherical shaped particles with the following physicochemical properties: mean particle size of 142.5 nm, polydispersity index of 0.195, the zeta potential of 􀀀 24.6 mV, and percentage entrapment efficiency of 90.46%. Also, the formulated NPs sustained drug release for up to 72 h. In-vitro cytotoxicity studies emphasized the superior anti-cancer activity of DDC-loaded NPs against the RCC cell line, compared to either pure or physical mixture of GT and 5FU. Furthermore, DDC-loaded NPs exerted a potent apoptotic response in the zebrafish embryos model, compared to free DDC or pure drugs. Collectively, our results suggest that the prepared DDC-loaded NPs might represent a promising therapeutic option for conquering renal carcinoma.