In this paper, we describe the development of a microfluidic culture-based biosensor for detecting mycobacteria in environmental samples. The biochips rely upon the unique paraffinophilic (i.e., wax-loving) nature of mycobacteria to rapidly and selectively differentiate them from non-target microorganisms. A new method of depositing and patterning paraffin was developed to fabricate the prototype biosensors using Opticlear as solvent to generate liquid paraffin and pattering with positive photoresist lift-off. The prototype biochips were experimentally tested to demonstrate the concept of rapid and selective detection of mycobacteria using pure cultures and epifluorescence microscopy to visualize microorganisms on the surface. Our successful demonstration of the culture-based biochip technology presents an alternative approach for developing new technology to track microorganisms in complex environmental samples. A microfluidic chip to selectively culture mycolic acid containing actinomycetes from environmental samples will improve screening for mycobacteria to protect public health as well as screening for mycobacteria to reduce the costs associated with treating nocardiafoam at sewage treatment plants.