Portable and low-cost analytical devices are essential for rapid detection of bioactive substrates in agricultural products. This study presents the first highly integrated microelectrochemical sensor based on pencil graphite for rapid and sensitive detection of hesperidin in Citrus reticulate ‘Chachi’ peel. The surface morphology and characterization as well as the electrochemical property of pencil graphite was investigated and discussed. A high electrocatalytic efficiency of hesperidin has been found at used pencil graphite-based microelectrodes. Kinetic analysis was carried out to further understand the electrochemical process of hesperidin at a pencil graphite microelectrode. Consequently, a portable and highly-integrated microelectrochemical sensor exhibits a sensitivity of 0.7251 μA cm−2 μM−1 and a detection limit as low as 25 nM (S/N = 3), and high selectivity was fabricated. Proposed microelectrochemical sensors were applied to electrochemically determinate the hesperidin content in the extract of Citrus reticulata “chachi” peel. As a result, the concentration of hesperidin in the actual real sample detected electrochemically with the proposed portable and low-cost microelectrochemical sensors is highly consistent to that obtained with a common chromatographic method, thus indicating the good reliability and that it can be used in practical applications.