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. Mycotoxins are secondary metabolites produced by filamentous fungi belonging to the genera Aspergillus and Penicillium 1. Mycotoxins are also found in animal derived foods such as milk due to intake of contaminated feed 2,3. Considering agricultural and economical aspects and possible implications on public health, the most relevant mycotoxins are aflatoxins, ochratoxins, fuminosins, T-2 toxin, and zearalenone (ZEA) 4. Aflatoxins pose a huge economic burden as they cause around 25% or more loss of the world's food crops every year 5. Afl M1 (4-hydroxy aflatoxin B1) and M2 (4-dihydroxy aflatoxin B1) have well proven carcinogenic and mutagenic potentiality and pose severe health consequences on milk consumers, including the risk of cancer and stunted growth in children below the age of 5 years 6. When B1 is ingested by a cow, it is secreted as hydroxylated metabolite aflatoxin M1 (Afl M1) in the urine and milk of the cow 7. Consumption of food containing aflatoxin concentrations of one milligram/kilogram or higher has been suspected to cause aflatoxicosis 8 , the prognosis of which consists of acute liver failure, jaundice, lethargy, and nausea, eventually leading to death within 1 to 2 weeks, based on past reports 9. Thus there is a need to develop rapid low cost technology based highly specific methods for detection of aflatoxins to improve surveillance and control in rural areas. A plethora of analytical techniques are available for aflatoxin M1 detection, ranging from chromatography and HPLC-MS used for regulatory control in official laboratories to rapid test kits for grain silos and farmers, especially for surveys when outbreaks occur 10,11. Conventional techniques for the detection of aflatoxins include gas chromatography (GC), fluorescence or UV based detection, thin layer chromatography (TLC) and enzyme linked immunosorbent assay (ELISA) 6-12. These techniques are routinely used and yield reliable results, however they are expensive, time consuming, require large scale instrumentation and large amounts of hazardous chemical reagents. Advancement in the development of bios...