•A volume miniaturized cyt c biosensor assay using screen printed electrode reported.•A new low-cost USB based electrochemical analyzer for cyt c analysis designed.•Implementation of GUI based system enables the analyzer easy to use.•Measurement of cyt c release in real samples also evaluated using the analyzer. In this paper, a portable, cost-effective electrochemical assay is presented for rapid, sensitive, and quantitative detection of cytochrome c (cyt c) release. The developed cyt c assay consists of two parts: (i) a miniaturized electrochemical biosensor based on cytochrome c reductase (CcR) functionalized screen printed electrodes (SPE); (ii) a microcontroller based data acquisition unit integrated with potentiostat circuit capable of performing cyclic voltammetry technique for the analysis. The working electrode surface of SPE was integrated with polypyrrole (PPy)-carbon nanotubes (CNT) nanocomposite for an enhanced immobilization of the enzyme, CcR. The acquired biosensor data are processed into digital form by the microcontroller and further transferred to a PC through USB port for analysis. GUI based system implemented here makes the analyzer easy to operate. Under optimal conditions, the electroanalytical behavior of the CcR-CNT-PPy-SPE biosensor linearly responds to the cyt c concentration range from 10nM to 500μM with a detection limit of 10nM and a sensitivity of 0.102±0.005μAμM−1cm−2. The performance of the volume miniaturized SPE based biosensor coupled with the portable microcontroller based instrument was further evaluated by applying it for the measurement of mitochondrial cyt c release during cardiomyocytes apoptosis; the results are validated well with the commercial electrochemical analyzer and standard ELISA.