3D printing of microfluidic lab-on-a-chip devices enables rapid prototyping of robust and complex structures. In this work, we designed and fabricated a 3D printed lab-on-a-chip device for fiber-based dual beam optical manipulation. The final 3D printed chip offers three key features, such as (1) an optimized fiber channel design for precise alignment of optical fibers, (2) an optically clear window to visualize the trapping region, and (3) a sample channel which facilitates hydrodynamic focusing of samples. A square zig–zag structure incorporated in the sample channel increases the number of particles at the trapping site and focuses the cells and particles during experiments when operating the chip at low Reynolds number. To evaluate the performance of the device for optical manipulation, we implemented on-chip, fiber-based optical trapping of different-sized microscopic particles and performed trap stiffness measurements. In addition, optical stretching of MCF-7 cells was successfully accomplished for the purpose of studying the effects of a cytochalasin metabolite, pyrichalasin H, on cell elasticity. We observed distinct changes in the deformability of single cells treated with pyrichalasin H compared to untreated cells. These results demonstrate that 3D printed microfluidic lab-on-a-chip devices offer a cost-effective and customizable platform for applications in optical manipulation.