Ochratoxin A (OTA), classified as a possible human renal carcinogen (group 2B), is a potent toxin as to cause the nephropathy. Many methods have been proposed and reviewed for OTA determination in food and agricultural products. However, current analytical procedures of mycotoxin are based on the time-delayed analysis. To reduce the contamination of OTA during distribution and storage of food and feeds, a rapid and easy-to-use detection method is required. The strip assay is an easy and fast detection method that is very reliable and cheap in production. The purpose of this study was to improve the sensitivity of strip sensor by simplifying the manufacturing steps and detection reading. Feasibility of strip assay detection of OTA was determined by color appearance of test line that was produced by the binding between OTA-BSA conjugates and gold antibody particles. However, in this study, strip assays were improved the efficacy of detection by conjugating with nanoparticles and OTA-BSA conjugates, instead of antibody. By different optimization steps in strip manufacturing and the application of the label on the strips, an increase in sensitivity and applicability was accomplished. The method uses a low cost test device consisting of a conjugation pad, membrane, sample pad, and absorbent pad. OTA-BSA and their conjugates with colloidal gold nanoparticles were prepared. The detection was based on the competition of OTA in a sample and an OTA-BSA on the colloidal particle surfaces for the binding to antibody of OTA immobilized on a membrane. It allows direct analysis of sample containing 10% methanol in phosphate buffered saline. The limit of detection obtained was 10 ng/ml for OTA. The cross reactivity of OTA strip assays with Aflatoxin B1 (AFB1) was examined. When 10, 100 ng/ml of AFB1 was tested, non-specific binding was not observed in the test strip.