As a result of their favorable physical and chemical characteristics, thermoplastics have garnered significant interest in the area of microfluidics. The moldable nature of these inexpensive polymers enables easy fabrication, while their durability and chemical stability allow for resistance to high shear stress conditions and functionalization, respectively. This review provides a comprehensive examination several commonly used thermoplastic polymers in the microfluidics space including poly(methyl methacrylate) (PMMA), cyclic olefin polymer (COP) and copolymer (COC), polycarbonates (PC), poly(ethylene terephthalate) (PET), polystyrene (PS), poly(ethylene glycol) (PEG), polylactic acid (PLA), acrylonitrile butadiene styrene (ABS), and polyester. We describe various biofunctionalization strategies applied within thermoplastic microfluidic platforms and their resultant applications. Lastly, emerging technologies with a focus on applying recently developed microfluidic and biofunctionalization strategies into thermoplastic systems are discussed. Display omitted •Physical attachment of biomolecules in thermoplastic microfluidic channels.•Robust immobilization of biomolecules via covalent bonds in thermoplastic polymers.•3D scaffolds on thermoplastic microfluidics.•Innovative ideas for bio-functionalization of thermoplastic microfluidics.