This study describes the colorimetric detection of aflatoxin M1 (Afl M1) in milk samples using a microfluidic paper-based analytical device (µPAD). Fabrication of µPADs was done using a simple and quick approach. Each μPAD contained a detection zone and a sample zone interconnected by microchannels. The colorimetric assay was developed using unmodified AuNPs as a probe and 21-mer aptamer as a recognition molecule. The free aptamers were adsorbed onto the surface of AuNPs in absence of Afl M1, even at high salt concentrations. The salt induced aggregation of specific aptamers occurred in presence of Afl M1. Under optimum conditions, the analytical linear range was found to be 1 µM to 1 pM with limit of detection 3 pM and 10 nM in standard buffer and spiked milk samples respectively. The proposed aptamer based colorimetric assay was repeatable, quick, selective, and can be used for on-site detection of other toxins in milk and meat samples.