We report in this work a one-step approach for the formation of self-assembled monolayers (SAMs) from thiourea (TU) over gold nanoparticles dispersed in carbon nanotubes (CNTs–Au_nano). The fabrication of the sensor is accomplished by drop-casting a thin film from an optimized mixture of CNTs, Au_nano, and TU over a GC electrode surface. The TU-SAMs electrochemical sensor is used for the simultaneous determination of dobutamine (DB), paracetamol (PA), amlodipine (AM), and daclatasvir (DAC), in human serum and in commercially available tablets. The proposed sensor is efficient, simple, and time saving in its preparation compared to other methods cited in the literature that need a longer time. The TU-SAMs sensor shows a high electrocatalytic effect for the simultaneous determination of DB, PA, AM, and DAC in human blood serum in concentration ranges 0.03 to 20 μM, 0.05 to 40 μM, 0.01 to 35 μM and 0.01 to 20 μM with low limits of detection of 0.85, 3.78, 0.65 and 0.60 nM respectively. The sensor shows good recoveries and resistance toward common interferents in human serum. The figures of merit such as stability, repeatability and robustness are presented.