Nowadays, the bipolar electrochemiluminescence (BP-ECL) is basically performed in complicated and expensive microfluidic devices, which may limit its wide applications. Here, a paper-based BP-ECL biosensor using "U"-shaped bipolar electrode (BPE) is developed for detection of oxidase-substrate biomarkers in human serum samples, with no need for complicated and expensive microfluidic technologies and peripheral apparatuses. The BP-ECL paper chips (PCs) can be well fabricated by two screen-printing processes: wax-screen-printing for on-paper hydrophilic and hydrophobic patterns, and carbon ink-screen-printing for BPEs and driving electrodes on wax-patterned paper. A low-cost and portable CCD is used to detect ECL signals from the luminol-H2O2 reaction at the BPE anodic pole. Under optimized conditions, the paper-based BP-ECL biosensor can measure the concentrations of H2O2, with a linear range of 5-10000 mu M and a detection limit of 0.424 mu M. Importantly, the biosensor is capable of achieving a linear range of 10-5000 mu M for choline, lactate or cholesterol; and the respectively corresponding detection limits are 0.573 11M, 3.132 mu M and 7.418 mu M. Moreover, it has an acceptable detection range, sensitivity, stability and selectivity. Finally, the developed method is successfully applied for determination of choline, lactate or cholesterol in human serum. (C) 2018 The Electrochemical Society.