In clinical practice, thymopentin (TP-5) is a commonly utilized immunomodulatory peptide drug. The relatively short half-life of TP-5, however, significantly limits its applicability in immunotherapy. Inspired by the structure of the TLR2 ligand lipopeptide Pam3CSK4, fatty acid-modified TP-5 peptides were designed and synthesized in this study. Utilizing its amphiphilicity, they were sonicated to assemble into nanoparticles with the diameters of approximately 100 nm. Compared with TP-5, TP-5 monopalmitate-modified nanoparticle has immune-activating properties both in vivo and in vitro. It markedly increased TNF-α secretion from RAW264.7 cells and aided in the maturation of DCs. The immunogenicity of OVA model antigen was increased in vivo when capsulated by TP-5 lipopeptide nanoparticle, which considerably slowed the growth of B16-OVA melanoma. This fatty acid-modified TP-5 assembled nanoparticle offers a straightforward and useful delivery system for the design of innovative nanovaccine for cancer immunotherapy.