With the development of biomedical technology, personalized diagnosis and treatment at the single-cell level are becoming more important in the medical field. As one of the most powerful tools, microfluidic chips have shown significant potential for various applications related to cell separation, cell proliferation, and cell behavior analysis. However, fabricating microfluidic devices requires complicated procedures and high-cost equipment. In this study, an optofluidic maskless lithography method was proposed for rapid fabrication of microfluidic devices integrated with microwells. Through the use of this approach, microwells can be on-line designed and the exposure patterns can be modulated. Single or multi polystyrene microspheres were successfully trapped by using the designed microwells. The capture of MCF-7 cells and cell arrays indicated that the microfluidic devices fabricated in the present study can be applied for cell research. © 2019 by the authors.