Gemcitabine, a chemotherapeutic agent widely approved for treating various cancers, faces significant clinical challenges, including drug resistance and poor tumor selectivity. To address the limitation of inadequate tumor targeting, a peptide chain was developed to specifically bind to colon cancer cells and conjugated to gemcitabine via aldehyde-amine condensation. This study aimed to exploit the oncogenic activity of the multifunctional protein α-Enolase (ENO1). The therapeutic efficacy of gemcitabine conjugated with the ENO1-targeting peptide (GCB-P) was evaluated through in vitro and in vivo experiments. Cytotoxicity assays and protein blotting analyses were performed on cell lines, including NCM-460, HCT116 and SW620. GCB-P exhibited significantly enhanced selectivity and potency against colon cancer cells compared to gemcitabine alone, with minimal cytotoxicity to normal colorectal cells. In addition, GCB-P demonstrated superior tumor-selective accumulation and release at the tumor site, as evidenced by in vivo metabolic analyses. These findings underscore the potential of GCB-P as an effective colon cancer treatment with reduced off-target toxicity. Furthermore, GCB-P displayed acid-responsive properties, facilitating precise delivery to tumor sites with the aid of homing peptides. These results highlight the promise of ENO1-targeted peptide modification for gemcitabine in developing targeted drug delivery systems for colon cancer.