In this study, we developed a new, efficient targeting and pH-responsive formulation using folate (FA)-conjugated human serum albumin (HSA) nanoparticles to deliver doxorubicin (DOX) drug to renal cell carcinoma. FA was conjugated to HSA, and DOX was loaded into obtained FA-HSA NP using the desolvation method. FA-HSA-DOX NPs were examined using zeta potential and (DLS) measurements, dynamic light scattering, scanning electron microscopy, atomic force microscopy, FTIR, and UV-Vis Spectroscopy. Drug liberation rate, toxicity/viability rate, and the apoptosis/necrosis effect of different DOX, FA-HSA, and FA-DOX-HSA treatments were evaluated using release profile study, MTT assay, and flow cytometry methods, respectively. The expression of BAK, BCL2, BAX, and BCL-XL genes in cancer cells after treatment was analyzed via real-time polymerase chain reaction (PCR). The average size, zeta potential, and polydispersity of FA-DOX-HSA NPs were 200 ± 9 (nm), − 47 ± 7 (mv), and 0.012, respectively. This formulation showed pH-dependent drug release, high drug loading performance, sustained ability of drug release. The notable toxic properties and high apoptosis-stimulation ability of the FA-HAS-DOX NPs in RCC-GH cell lines demonstrated long-term anticancer efficacy. Also, real-time PCR results showed an increase in BAX expression and a decrease in BCL-2 L1 and BCL-XL significantly. Our findings indicate that FA-HSA NPs have no cytotoxic effect on cancer cells (RCC-GH) and healthy cells (RPTEC/TERT1). Our study provides a simple and efficient FA-HSA carrier with intrinsic biocompatibility for DOX drug delivery