BACKGROUND: Acquired immune deficiency syndrome (HIV/AIDS) has been a major global health concern for over 38 years. No safe and effective preventive or therapeutic vaccine has been developed although many products have been investigated. Computational methods have facilitated vaccine developments in recent decades. Among HIV-1 proteins, p24 and Nef are two suitable targets to provoke the cellular immune response. However, the fusion form of these two proteins has not been analyzed in silico yet. OBJECTIVE: This study aimed at the evaluation of possible fusion forms of p24 and Nef in order to achieve a potential therapeutic subunit vaccine against HIV-1. METHOD: In this study, various computational approaches have been applied to predict the most effective fusion form of p24-Nef including CTL (Cytotoxic T lymphocytes) response, immunogenicity, conservation and population coverage. Moreover, binding to MHC (Major histocompatibility complex) molecules was assessed in both human and BALB/c. RESULTS: After analyzing six possible fusion protein forms using AAY linker, we came up with the most practical form of p24 from 80 to 231 and Nef from 120 to 150 regions (according to their reference sequence of HXB2 strain) using an AAY linker, based on their peptides affinity to MHC molecules which are located in a conserved region among different virus clades. The selected fusion protein contains seventeen MHC I antigenic epitopes, among them KRWIILGLN, YKRWIILGL, DIAGTTSTL and FPDWQNYTP are fully conserved between the virus clades. Furthermore, analyzed class I CTL epitopes showed greater affinity binding to HLA-B 5701, HLA-B51:01 and HLA-B 27*02 molecules. The population coverage with the rate of >70% coverage in the Persian population supports this truncated form as an appropriate candidate against HIV-I virus. CONCLUSION: The predicted fusion protein, p24-AAY-Nef in a truncated form with a high rate of T cell epitopes and high conservancy rate among different clades, provides a helpful model for developing a therapeutic vaccine candidate against HIV-1.