Insulin hormone is of great importance for many diseases, especially for diabetes management. Therefore, different detection strategies have been used for sensitive and fast detection of insulin in physiological conditions. In this study, an electrochemical signal switch aptasensor for sensitive detection of insulin has been developed based on methylene blue (MB)–modified insulin-specific aptamer immobilized on gold-deposited screen-printed electrodes. The aptamer fabrication parameters of aptamer, blocker and insulin incubation times, were optimized using the response surface method as 180 min, 60 min, and 25 min, respectively. Optimized values were then used to fabricate aptasensor, and analytical parameters were calculated using square wave voltammetry. The calibration of the aptasensor was performed based on the current difference calculated by subtracting the respective current values obtained for the MB-probe’s on and off positions. The linear working range and limit of detection of the aptasensor were calculated as 25–150 pM and 18.5 pM, respectively. The relative standard error and accuracy of the aptasensor were 9.5% and 6.4%, respectively. The interference study showed no significant interfering substances except for uric acid, and the stability of the sensor was good for 10 days keeping 92% of its initial performance. The developed aptasensor showed promising results for easy and sensitive insulin detection in physiological conditions.