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The development of in vitro models that mimic the immunological, mechanical, and structural properties of the gut, as well as its crucial microbial symbionts, facilitate the real-time assessment of host-microbiome interactions. Here, we describe our development of a threespine stickleback fish “gut-on-chip” biomimetic microdevice which will co-culture complex microbial communities with mucus-secreting intestinal epithelia and leukocytes. This chip was fabricated using soft lithography with liquid PDMS and consists of a middle channel coated with extracellular matrix (ECM) compounds separating two microfluidic side channels. After temperature cure, PDMS is bound on a glass slide by plasma activation, forming a closed microenvironment where cells can be grown. We have successfully isolated and independently cultured stickleback intestinal epithelial cells and leukocytes and evaluated their tolerance to various components of ECM. Our results show that fibrinogen is more suitable for culturing intestinal leukocytes compared to collagen-1 or gelatin. Leukocyte viability is more sustainable after stimulation with LPS-B5 compared to unstimulated. Unlike immune cells, stickleback epithelial cells do not survive on fibrinogen and show better viability on the collagen-1 coated medium. This study establishes the unique culture conditions necessary for teleost microfluidic gut-on-a-chip and will advance our understanding of the host-microbiome interactions in this non-model system. Supported by  Gordon and Betty More Foundation, SASI Symbiosis Model Systems #9323