Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the second in females, whose survival ratio and indicating biomarkers are limited. The rapid development of multiple immunofluorescences gives rise to widespread applications of this new advanced technology called multiplex immunohistochemistry (mIHC), which makes it possible to detect several fluorescent proteins on the same tumor tissue microarray (TMA) within the same time and spatial organization. By taking advantage of this mIHC technology, we detected three tumor-associated antigens (TAA) including the human epidermal growth factor receptor 2 (HER2), the cluster of differentiation 133 (CD133), the programmed death ligand-1 (PD-L1), and one immune-associated macrophage marker, the cluster of differentiation 68 (CD68) in cancer tissues versus para-carcinomatous normal tissues derived from a cohort of 84 CRC patients. All four markers were upregulated in cancer tissue compared with normal tissues. And the expressions of CD133, HER2, PD-L1, and CD68 were correlated with pathological grade, T stage, tumor size, metastasis, respectively. Accordingly, CD133 and PD-L1 could be applied as potential diagnostic biomarkers for CRC at an early stage, while the enrichment of HER2 might act as an advanced indicator in aggressive cancer status of CRC; whereas, CD68 could be potentially considered as an advanced diagnostic indicator in CRC patients, as well as a metastatic promoter in CRC-related TME. The differential expression of these four proteins, as well as their clinicopathological correlation, indicates that these four proteins could be utilized as specific diagnostic and prognostic biomarkers in CRC patients.