An on-chip enzyme-linked immunosorbent assay combined with an electrochemical detection method (EC-ELISA) was employed to detect a leptin, one of the most important adipose derived hormones, using gold electrodes modified with a tri(ethylene glycol) terminated short alkanethiol (TEGCnSH, Cn = (CH(2))n, n = 2, 4, 6, and 8) monolayer. These TEGCnSH monolayers on gold electrodes can suppress non-specific protein adsorption without affecting the electrochemical activity required for detecting p-aminophenol (PAP), which is an alkaline phosphatase (ALP) product. We measured leptin with a highly sensitive detection range (100 pg mL(-1) to 10 ng mL(-1) level) and with the desired detection limit (13.6 pg mL(-1)) by using electrochemical detection. For detecting leptin, the EC-ELISA method using TEGC4SH modified gold electrode with a poly(dimethylsiloxane) based microchannel was superior to the conventional ELISA method. With the EC-ELISA method, we were able to measure leptin with a satisfactory detection range and a pg level detection limit within 30 min, which is a much lower detection level than that obtained with conventional plate based ELISA.