This article presents an improved method for the three-dimensional cultivation of colon cancer cells and the realization of real-time fluorescence imaging using a microfluidic chip. By incorporating endogenous red fluorescent protein into the colon cancer cells, we employed laser confocal microscopy to visualize the cells cultivated within the chip in three dimensions. The expression of intracellular red fluorescent protein allowed for the observation of cell growth status, facilitating real-time monitoring and high-resolution fluorescence imaging. Moreover, immunofluorescence staining was employed to characterize feature proteins indicative of cellular activity, with their fluorescence intensity demonstrating a positive correlation with protein expression. The research findings indicate that the expression of activity-related proteins is influenced by the microenvironment, exhibiting stronger activity in the three-dimensional cultivation on the chip compared to the two-dimensional approach. This suggests that the microenvironment within the chip more closely resembles the actual microenvironment within the human body. This method provides a novel technical approach and experimental platform for further exploration of tumor metastasis mechanisms and the screening of related drugs.