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We report a new two-axis active optical fiber manipulator for on-chip optical manipulation and detection in microfluidic environment. The system comprising of air chambers, fiber channels, controllable moving walls, and membrane structures were fabricated by using microelectromechanical systems technology. By adjusting air pressures to control the deflection of the pneumatic chambers placed orthogonal to and underneath the fiber channels, accurate alignment of a pair of approximately coaxial optical fibers, which was indicated by maximizing fiber-to-fiber optical-coupling measured in real time, has been achieved. A maximum displacement of a buried fiber as large as 13 mum at an applied pressure of 40 lb/in2 for one air chamber has been demonstrated. It was sufficient to accurately align two approximately coaxial optical fibers to maximize the optical coupling efficiency. The maximum coupling efficiency for two single-mode optical fibers facing each other at a distance of 200 mum was measured to be 4.1%. The following features have been successfully demonstrated with this system: 1) stable optical trapping and stretching of a single red blood cell; 2) stable optical trapping of multiple microparticles; 3) optically driven controlled motion of single and multiple microparticles; and 4) integration of a counterpropagating dual-beam trap with single-beam optical tweezers. In addition to optical trapping and manipulation, the proposed device is promising for applications requiring coaxial input/output fibers for in-line optical analysis. Furthermore, it can be easily integrated with other microfluidic devices such as microcapillary electrophoresis channels or microflow cytometers for DNA, protein, and cell analysis.