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A leakage-free and disposable biochip for deoxyribonucleic acid (DNA) separation and detection is developed in this study. The biochip comprises of 50 mm long polydimethylsiloxane (PDMS) microchannel and copper electrodes engraved on flame retardant (FR-4) printed circuit board (PCB) substrate. An inhibitor made from photocurable diacrylate bisphenol-A polymer (DABA) was used to establish a permanent bonding between PDMS and PCB substrates. Pull-off test experiments resulted in average strength of 287.4 kPa and a standard deviation of ± 23.8 kPa. These results are comparable to recent studies on leak-free microfluidic applications. Meanwhile, the leakage test showed that the biochip could withstand a pressure of more than 190 kPa, which is sufficiently high for most DNA measurements. Finally, experiments performed on two different DNA samples indicated that the proposed design could accurately detect DNA fragments with current sensitivity higher than 100 nA, under electric field strength of 20 V/cm. The new design effectively seals the biochip, thus preventing leakage of liquid from the sensor matrix. Together with its biologically and electrochemically inert characteristic, it opens up possibilities in building a truly portable biosensing device.