Biosensors based on ion-sensitive field effect transistors (ISFETs) combined with aptamers offer a promising and convenient solution for point-of-care testing applications due to the ability for fast and label-free detection of a wide range of biomarkers. Mobile and easy-to-use readout devices for the ISFET aptasensors would contribute to further development of the field. In this paper, the development of a portable PC-controlled device for detecting aptamer-target interactions using ISFETs is described. The device assembly allows selective modification of individual ISFETs with different oligonucleotides. TaO-gated ISFET structures were optimized to minimize trapped charge and capacitive attenuation. Integrated CMOS readout circuits with linear transfer function were used to minimize the distortion of the original ISFET signal. An external analog signal digitizer with constant voltage and superimposed high-frequency sine wave reference voltage capabilities was designed to increase sensitivity when reading ISFET signals. The device performance was demonstrated with the aptamer-driven detection of troponin I in both reference voltage setting modes. The sine wave reference voltage measurement method reduced the level of drift over time and enabled a lowering of the minimum detectable analyte concentration. In this mode (constant voltage 2.4 V and 10 kHz 0.1Vp-p), the device allowed the detection of troponin I with a limit of detection of 3.27 ng/mL. Discrimination of acute myocardial infarction was demonstrated with the developed device. The ISFET device provides a platform for the multiplexed detection of different biomarkers in point-of-care testing.