In this study, an electrochemical and aptamer-based aptasensor was developed for the sensitive detection of patulin, a mycotoxin commonly found in fruits and fruit-based products. The aptasensor used an innovative structural switching signal-off platform for detecting patulin. The aptamer immobilization on screen-printed carbon electrodes was achieved through Au electrodeposition and thiol group (-SH) route. Response surface methodology was used to determine the optimal incubation times for the aptamer, blocking agent, and target molecule, which were found to be 180 min, 40 min, and 89 min, respectively. The response of the aptamer to different concentrations of patulin was measured using square wave voltammetry by exploiting the structural switching mechanism. The sensor response was determined by quantifying differences in the aptasensor's background current. The aptasensor exhibited a linear working range of 1–25 μM and a low detection limit of 3.56 ng/mL for patulin. The aptasensor's relative standard deviation and accuracy were determined to be 0.067 and 94.4%, respectively. A non-specific interaction was observed at low concentrations of two other mycotoxins, ochratoxin A and zearalenone. The interference from ochratoxin A in the measurements was below 10%. In real sample tests using apple juice, interference, particularly at low concentrations, had changed the recovery of patulin negatively with a significant effect on the structural switching behavior. Nevertheless, at a concentration of 25 ng/mL, the interference effect was eliminated, and the recovery standard deviation improved to 6.6%. The aptasensor's stability was evaluated over 10 days, and it demonstrated good performance, retaining 13.12% of its initial response. These findings demonstrate the potential of the developed electrochemical aptasensor for the sensitive detection of patulin in fruit-based products, with prospects for application in food safety and quality control.