Enzyme-linked immunosorbent assay (ELISA) is one of the most important technologies for biochemical testing critical for diagnosis and monitoring of many diseases. Traditional systems for ELISA incubation and reading are expensive and bulky, thus cannot be used at point-of-care or in the field. Here, we designed and demonstrated a new miniature mobile phone-based system for ELISA. This mHealth system can be used to complete all steps of the assay, including incubation and reading. It can be fabricated at low cost, it is portable, and can transfer test results via mobile phone. We have designed incubation chamber, imaging enclosure, and data processing algorithm. We demonstrate how mobile ELISA can be calibrated for accurate measurements of cortisol and show successful measurements with the calibrated system. We show that the results acquired with our prototype match well the results obtained with the gold standard.