A simple electrochemical sensor based on electrochemically synthesized Fe3O4 nanoparticles was constructed by an ink with the nanoparticles, isopropanol, NAFION and carbon Vulcan to detect dopamine, uric acid and ascorbic acid. The electrocatalytic activity of the nanoparticles for the oxidation of the analyte molecules was examined by means of cyclic voltammetry and square wave voltammetry. The parameters controlling the performance of the sensor were optimized, such as the amount of Fe3O4 nanoparticles (1, 2, 3, 5, 8, 10 mg), amount of binder (5, 10, 15 µL) and carbon Vulcan in the ink (4, 6, 8 mg). The temperature was maintained at 25 °C and the pH was 7.5 with buffer phosphate. The optimal sensor conditions were 8 mg magnetite, 4 mg carbon Vulcan and 5 µL of NAFION@ 117. The calibration curves for the three analytes were determined separately, obtaining linear ranges of 10–100, 20–160 and 1050–2300 µM and limits of detection of 4.5, 14 and 95 µM for dopamine, uric acid and ascorbic acid, respectively. This electrochemical sensor has also shown significant sensitivity and selectivity without interference from the three analyte molecules presented simultaneously in solution. This sensor was applied for the detection of these molecules in real samples.