Colorectal cancer (CRC) remains one of the most common malignancies and a major cause of cancer mortality. In this study, novel tumor antigens were screened from induced pluripotent stem cells (iPSCs) and evaluated through a series of in vitro immunological assays to identify the optimal antigen/adjuvant vaccine combination. Using the NetMHCcons platform developed by DTU Health Tech and Western blot analysis, the tumor-associated antigen Nucleolin (NCL) derived from murine iPSCs was identified as a promising CRC vaccine candidate. Flow-cytometric dendritic-cell (DC) activation assays suggested that NCL could enhance the activation of cytotoxic CD8⁺ T cells. Moreover, NCL was tested in combination with various Toll-like receptor (TLR) family adjuvants in cellular fluorescence imaging experiments. The results demonstrated that NCL effectively activated CD8⁺ T cells and exhibited significant anti-tumor activity against murine CRC cells, with the NCL/Poly(I:C) combination showing the strongest effect. Using the Nanolive CX-96 Focus holotomographic live-cell imaging system, direct anti-tumor effects of NCL were further observed. Finally, ELISA analyses were performed to explore the underlying anti-cancer mechanisms. Collectively, these findings indicate that NCL is a promising antigen candidate for CRC vaccine development. Future studies will focus on screening additional iPSC-derived neoantigens and advancing toward multi-antigen vaccine design and clinical applicability in humans.