Cell-imprinted polymer (CIP) based optical biosensors have transformed point-of-care detection. However, challenges remain in their portability and detection sensitivity, time, and cost. Herein, we present an imprinted polymer-based low-cost microfluidic device integrated into a portable enclosure that enables rapid and sensitive bacteria detection in real water. A portable 3D-printed platform was custom-designed, housing all essential detection components, i.e., pumping and fluorescent imaging units and the microfluidic sensor. CIP coated magnetic microparticles (MPs) with affinity to bacteria were manipulated inside the magnetophoretic microfluidic device at an optimized flow rate of 0.01 mL/min for bacteria capturing. Fluorescent imaging pre- and post-bacteria capture facilitated quantification of fluorescence intensity changes as bacteria were trapped by the CIP-MPs. The sensor’s dose–response curve established limits of detection (LOD) and quantification (LOQ) at 8 × 103 and 6 × 105 CFU/mL, respectively, within a dynamic range of 103 to 109 CFU/mL. It specifically detected E. coli, distinguishing it from non-specific bacteria like Salmonella and Sarcina. In real pond water tests, our sensor detected 2 × 10⁶ CFU/mL, matching a central lab’s result of 2.33 × 10⁶ CFU/mL, demonstrating its effectiveness for real-water monitoring. While further enhancements are needed for improving the specificity in complex environmental matrices and broader bacterial strain detection, the sensor’s simplicity and portability highlight its potential for practical potential.