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This paper reports on design and rapid constructions of microstructures, such as in-fiber microhole, D-shaped fiber, in-fiber microchannel, side-polished fiber and tapered fiber, in optical fibers using a CO2 laser system that help exposing the optical fiber core to the measurands. Exposing the optical fiber core to the measurands surrounding the fiber is often used to enhance the sensitivity of an optical fiber sensor. The microstructures in the fibers were examined using a SEM and an optical microscope. Testing on an in-fiber microchannel of 100 mum width, 210 mum depth and 10 mm length for fluorescence sensor embedded in a microfluidic card shows the prospect of this method for use as a fluorescence biosensor in a microfluidic card. The microstructures were fabricated using a direct-write continuous wave (CW) CO2 laser system with maximum output power of 40 W. The direct-write CO2 laser machine is a flexible and fast machining tool for design and fabrication of microfluidic cards and microstructures in an optical fiber, and can possibly be a replacement of the time consuming chemical etching method that is usually used for microstructure fabrications in an optical fiber.