Abstract: At a single snapshot without any mechanical movement, real-time compact multispectral imaging (MSI) is capable of capturing multiple 2-dimensional (XY) images that are corresponding to different characteristic spectrums () of the target. Real-time compact MSI has desirable features including high efficiency, robust to vibration, and a small volume. A two-dimensional narrow band multichannel micro-filter array (2DNBMFA) is an essential part to apply real-time compact MSI to in-vivo optical pathological diagnostic fluorescent imaging, and ultimately result in a real-time compact multispectral fluorescent imaging technology (MSFI). Firstly, the structural pattern was designed and the important technical parameters of the 2DNBMFA were determined, which was the key element to be used in the real-time compact MSFI. Secondly, the 2DNBMFA was manufactured which had a 2-dimensional spatial resolution and a high optical density. Experimental results indicate that the developed micro-filter array has a spatial resolution of 52 m52 m, full-width at half-maximum of 24 nm, optical density of 4, weak cross-talk between different optical channels. All these suggest that the real-time compact MSFI technology is technically feasible, which has the potential to capture the 2-dimensinal distribution of multiple fluorescent probes at any instant time. The real-time compact MSFI, if successfully be developed, would have great significance in medical in-vivo diagnostic imaging and in biological research area where it is hot to use multiple fluorescent probes as biological markers for a better understanding of various life processes.