Fluorescence microscopy imaging of live cells has provided consistent monitoring of dynamic cellular activities and interactions. However, because current live-cell imaging systems are limited in their adaptability, portable cell imaging systems have been adapted by a variety of strategies, including miniaturized fluorescence microscopy. Here, we provide a protocol for the construction and operational process of miniaturized modular-array fluorescence microscopy (MAM). The MAM system is built in a portable size (15c m×15c m×3c m) and provides in situ cell imaging inside an incubator with a subcellular lateral resolution (∼3µm). We demonstrated the improved stability of the MAM system with fluorescent targets and live HeLa cells, enabling long-term imaging for 12 h without the need for external support or post-processing. We believe the protocol could guide scientists to construct a compact portable fluorescence imaging system and perform time-lapse in situ single-cell imaging and analysis.