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Purpose: Plasma leptin is secreted from adipose tissues and plays pivotal roles in human physiological and pathological processes. Here, we aimed at conducting a protein biochip-based sandwich-like approach for detection of plasma leptin among healthy individuals, obesity, and diabetes patients., Experimental Design: Totally, 96 plasma samples, including 45 healthy individuals with standard body mass index (BMI), 28 obesity and 23 diabetes patients, were recruited in the study. Plasma leptin was detected by a well-established protein biochip. Meanwhile an ELISA was also performed for assessment of the leptin detection by the protein biochip., Results: We found that the plasma leptin level in the obesity and diabetes patients was significantly higher than that in healthy individuals with standard body mass index (p < 0.001). The limit detection concentration of leptin was as low as 0.006 μg/mL. The plasma leptin could be semiquantitatively detected by the protein biochip. The compatibility of the biochip-based detection approach seemed acceptable in comparison with the ELISA assay (R 2 = 0.948)., Conclusions: We provided a protein biochip-based approach for plasma detection. This approach would be a potential substitution for the ELISA assay., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)