Sensitive detection of methimazole (MMI) at the femtoliter (fL) scale has been achieved based on photo thermal lensing of gold nanoparticles (GNPs). GNPs were used because their surface plasmon resonance (SPR) have absorption in visible range. A combined glass microfluidic chip-photo thermal lens microscopy (MFC-PTLM) system with appropriate laser source was assembled for detection of MMI with GNPs. The thiol group of MMI bound to the surface of GNPs and created the core-shell form and reduced the surface plasmon resonance of GNPs as a consequence the PTLM signal of GNPs decreased following the increase of MMI concentration. The results showed that the changes of PTLM signal in the focal volume of 2.68 fL were proportional to the concentration of MMI over the range of 75-900 × 10-9 mol L-1. The obtained detection limit of this method was 46.5 × 10-9 mol L-1 and the relative standard deviation for ten repeated measurements of 550 × 10-9 mol L-1 MMI solutions was 3.47%. The developed method was successfully applied for determination of MMI in human serum and pharmaceutical samples.