As an advanced cell culture platform, the organ-on-a-chip has been in the spotlight recently owing to its ability to maintain the physiological characteristics of cells in vitro. Therefore, several disease models have been developed using the organon-a-chip technology, and the organ specific three-dimensional (3D) structure and various mechanical/chemical stimuli built into the chip enable efficient development of drugs, medical devices, and biomaterials, as well as realization of patientspecific precise medicine. This study introduces a novel chip-based non-muscle invasive bladder cancer model, multilayered tissue-on-a-chip (MToC), which was created using 3D bio-printing technology, micro-milling, and soft lithography based polydimethylsiloxane (PDMS) casting. All types of cells, T24, MRC-5, and HUVEC, were successfully co-cultured in the MToC. Using computational fluid dynamics (CFD), the flow phenomena occurring in MToC were analyzed. Further, we attempted Bacillus Calmette–Guérin (BCG)-induced migration of THP-1, and the viability reduction of bladder cancer cells and the THP-1 migration were observed. Although follow-up studies are needed to precisely mimic the immune response, this partial phenomenon of the immune response suggests the potential of this device as a surrogate experimental tool for BCG immunotherapy in future.