Microfluidic devices are now the most promising tool to mimic in vivo like scenarios such as tumorigenesis and metastasis due to its ability to more closely mimic cell’s natural microenvironment (such as three dimensional (3D) environment and continuous perfusion of nutrients). In this study, the ability of 2% and 3% enzymatically-crosslinked silk fibroin (eSF) hydrogels with different mechanical properties were tested in terms of colorectal cancer cell migration, under different microenvironments in a 3D dynamic model. Matrigel was used as control. Moreover, we present a comprehensive comparison between the traditional Boyden chamber assay and our 3D dynamic microfluidic model in terms of colorectal cancer cell migration. Our results show profound differences between the two used biomaterials and the two migration models, which were explored in terms of mechanical properties of the hydrogels as well as the intrinsic characteristics of the models. Moreover, we validated the developed 3D dynamic model by demonstrating that hVCAM-1 plays a major role in the extravasation process, influencing extravasation rate and traveled distance. Furthermore, the developed model enables precise visualization of cancer cell migration within a 3D matrix in response to microenvironmental cues, shedding light on the importance of biophysical properties in cell behavior.