In the tumor microenvironment, cancer cells create conditions characterized by nutrient depletion, waste accumulation, acidification, and hypoxia. These environmental factors inhibit various immune cell functions, including the ability of natural killer (NK) cells to target and eliminate cancer cells. The mechanisms governing the interaction between immune and tumor cells within the tumor microenvironment remain poorly understood. In this study, we aimed to describe the in vivo tumor cell behavior using microfluidic chip technology, which allowed us to create dynamic and hypoxic conditions. Specifically, we focused on the migration of NK cells. We formed tumor spheroids within a collagen matrix and created an adjacent channel where NK cells were present. We observed that the oxygen concentrations ranged from 1.07% to 6.25% at a distance from the scavenging line post 1 hour and from 0.08% to 5.68% post 150 minutes. The average oxygen concentration within the chip remained < 5.03% over a 24-hour period. Using an in vitro microfluidic tumor chip, we assessed how the activity of NK cells is altered in an immunosuppressive environment such as low oxygen, mimicking tumor hypoxia. We induced NK cell migration under low oxygen and dynamic conditions of 400 μL/hour in a cancer-NK cell model for 24 hours. We observed a 9.4-fold increase in NK cell migration into the collagen matrix under hypoxia compared to that under normoxic conditions. Additionally, through gene expression analysis of each model, we confirmed a significant 17.2-fold increase in the expression of CXC motif chemokine ligand 9 (CXCL9) in HepG2 cells exposed to low-oxygen conditions. This study demonstrated that low-oxygen conditions enhanced CXCL9 expression in HepG2 cells and promoted the migration of NK cells in vitro.