Aflatoxin B1 (AFB1) is the most toxic contaminant, causes carcinogenic, and immune toxic effects. There is an immense need to develop inexpensive, reliable biosensors for AFB1. The electrochemical aptasensor for detection of aflatoxin B1 has received much attention due to their trustworthy procedure. In this work, we have designed an electrochemical aptasensor by using methylene blue (MB) as a signalling fragment and polythiophene-3-carboxylic acid (PT3C) as the signal-enlarging platform. The (PT3C) was first screened onto the screen printed carbon electrode (SPCE) interface, and then the MB-tagged aptamer was immobilized on SPCE by using hexamethylenediamine (HMDA) as a spacer via carbodiimide amide-bonding chemistry. AFB1 has been chosen as model analyte to test the proposed aptasensing platform. A good dynamic range 2.5–30 ng L−1 was obtained for AFB1 with a limit of detection (LOD 1.6 ng L−1). The good reproducibility was recorded with RSD% of 3.21. The obtained straight-line equation was 3.6099 x + 19.073, R2 = 0.9977. For real-world sample applications, this aptasensor has been used in coffee samples. It displayed good recovery in the range 88.2–93.3%, thus showed the effectiveness of the proposed aptasensor for coffee samples. © 2019