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We present a straightforward method for fabricating functional micro-, submicro-, and nano-channels embedded in a porous carbon film and the application of this microfluidic platform for electrochemical sensing. A skin layer of poly(methyl methacrylate) (PMMA) fibers was first electrospun on a thoroughly cleaned Si wafer substrate, followed by spin coating of a polyacrylonitrile (PAN) film on top of the skin. High-temperature carbonization of the composite film decomposed the PMMA fibers and produced embedded microchannels in the PAN-derived amorphous monolithic carbon electrode. The channels were decorated with Pt nanoparticles by in situ thermal decomposition of a precursor metal salt to enhance functionality of the carbon electrode. Carbon electrodes decorated with Pt nanoparticles (carbon-Pt), with and without embedded microchannels, were used to detect the food toxin aflatoxin B1 (AFB1) by surface biofunctionalization with the antibodies of AFB1 (anti-AFB1) via an electrochemical impedance technique. The aligned nanochannels in the porous carbon film act as a reaction chamber for antigen–antibody interactions and provide channels for fast electron transport toward the electrode from the electrolyte, resulting in improved electrochemical performance of the biosensor. The fabricated immunosensor is highly sensitive to 1 pg/mL of AFB1 with a concentration range of 10 −12 –10 −7 g/mL. [ABSTRACT FROM AUTHOR]