Simple, novel, and direct detection of clinically important biomolecules have continuous demand among scientific community as well as in market. Here, we report the first direct detection and facile fabrication of a cysteine-responsive, film-based, self-powered device. NH2 functionalized BaTiO3 nanoparticles (BT-NH2 NPs) suspended in a three-dimensional matrix of an agarose (Ag) film, were used for cysteine detection. BaTiO3 nanoparticles (BT NPs) semiconducting as well as piezoelectric properties were harnessed in this study. The changes in surface charge properties of the film with respect to cysteine concentrations were determined using a current–voltage (I-V) technique. The current response increased with cysteine concentration (linear concentration range=10 µM–1mM). Based on the properties of the composite (BT/Ag), we created a self-powered cysteine sensor in which the output voltage from a piezoelectric nanogenerator was used to drive the sensor. The potential drop across the sensor was measured as a function of cysteine concentrations. Real-time analysis of sensor performance was carried out on urine samples by non-invasive method. This novel sensor demonstrated good selectivity, linear concentration range and detection limit of 10 µM; acceptable for routine analysis. © 2016 Elsevier B.V.