Parafilm ® is a thermoplastic film extensively used in laboratories for sealing or protecting flasks or cuvettes. In this paper, we report a simple, low-cost, biocompatible and detachable microfluidic chip incorporating easily accessible polymethylmethacrylate (PMMA), PVC and glass slides as the substrate materials, along with Parafilm ® as the bonding material. Multi-layer microfluidic chips can be fabricated by laser ablation followed by thermal fusion of constituent layers. A CO2 laser ablation method was employed to cut substrates as well as the Parafilm ® to obtain desired patterns. A thermal fusion bonding method was developed to bond the substrates in a single step. The patterned Parafilm ® was sandwiched in the PMMA, PVC or glass layers, and heating of the assembly with the addition of a static pressure, various functional microfluidic elements including microvalves, micropumps, and bioreactors were demonstrated. With Parafilm ® serving as a flexible membrane integrated to a fluidic channel, it was possible to implement a microvalve. A peristaltic micropump consisting of a sequence of interconnected gas-actuated microvalves was also fabricated. In addition, the biocompatibility of the PLT (Parafilm ® based laser ablation and thermal fusion bonding fabrication method) fabricated bioreactor was validated by culturing GFP-expressing E. coli, results showed bacterium were able maintain growth in a 7-day post-cultivation. In summary, the reported fabrication scheme, offers an inexpensive and versatile alternative for rapid prototyping of common microfluidic devices.