This work presents a novel, fast response time, plastic optic fiber (POF) biosensor to detect Escherichia coli. It discloses the technique for the development, calibration and measurement of this robust and simple-to-construct POF biosensor. The probes in U-shaped format were manufactured with a specially developed device. The calibration process led to the evaluation of the sensitivity, accuracy and repeatability by using solutions of sucrose for obtaining refractive indices (RI) in the range 1.33-1.39 IR equivalent of water and bacteria, respectively. The POF probes were functionalized with antibody anti-E. coli serotype O55 and tested firstly with saline and then with bacterial concentrations of 104, 106, and 108 colony forming units/ml (CFU/ml). The optoelectronic setup consists of an 880nm LED connected to the U-shaped probe driven by a sine waveform generated by the Simulink (from Matlab®). On the other side of the probe a photodetector generates a photocurrent which is amplified by a transconductance amplifier. The output voltage signal is read by the analog-to-digital (A/D) input of the microcontroller. In all tested concentrations, the results presented a tendency of a decrease in the output signal with time, due to the attachment of the bacteria to the POF probe and consequent increase in the RI close to the sensitive area of the fiber surface. It has been shown that the system is capable of providing positive response to the bacterial concentration in less than 10min, demonstrating good possibilities to be commercially developed as a portable field sensor. © 2013 Elsevier B.V.