Proteins play crucial roles in biological activities and show close relationship with human diseases as biomarkers. Thus, rapid, sensitive, selective, and portable detection of protein is in high demand for point-of-care (POC) testing. Here, a smartphone-controlled biosensor system is developed with electrochemical impedance spectroscopy (EIS) to detect proteins for POC testing. The system was constituted by miniaturized biosensor, hand-held EIS detector, and smartphone, collectively. Printed carbon electrodes and interdigital gold electrodes, were modified with bio-components and integrated into the system as biosensors for protein detection. Then, detections of bull serum albumin (BSA) and thrombin demonstrated analytical performances of the system for quantification of the protein binding and enzyme activities by special antibodies and peptides immobilized on the electrodes. By the impedance measurement, the system could detect BSA and thrombin as low as 1.78 μg/ml and 2.97 ng/ml, respectively. The results suggested that the smartphone-controlled biosensor system provided a portable and cost-effective platform that allowed integration of diverse biosensor formats for different kinds of protein quantifications for POC testing.