The presence of secondary metabolite, particularly aflatoxin produced by fungi, poses economic challenges in food and feed industries. This study aimed to identify aflatoxigenic Aspergillus species inedible oil and quantify total aflatoxins levels. We collected thirty sample of edible oil from various local market in Karachi and tested them for presence of Aspergillus flavus and total aflatoxin levels. Out of thirty, four samples tested positive for Aspergillus flavus using culture-based methods, which were subsequently confirmed by amplification of partial transcribed (ITS) region. Aflatoxin production was assessed using colorimetric methods, revealing that all A. flavus positive isolates (JB2, MGM4, LMM1, and RLM2) exhibited a plum red change upon exposure to ammonium hydroxide vapors on Sabouraud Dextrose Agar. Notably, JB2 also demonstrated color change on Yeast extract Sucrose and Coconut Agar medium, and exhibited blue fluorescence under UV light on coconut Agar medium. Aflatoxins were extracted from edible oil sample using solvent extraction method and quantified using a VICAM fluorometer. Among samples one exhibited total aflatoxin levels between 20-30ppb, five samples had levels below 20ppb, while 24 samples were negative for aflatoxin. These finding underscore the need of continued monitoring of aflatoxin contamination in edible oil.