To achieve simultaneous analysis of the three-dimensional spatial structure and spectral information of a target sample, a multi-spectral Gabor in-line digital holographic microscopy imaging system is proposed. Specifically, an adjustable multi-wavelength LED illumination source is introduced into the Gabor coaxial digital holographic imaging system. Holographic images of a target sample at different wavelengths are acquired, and the reconstructed images of the target sample at various wavelengths are obtained through angular spectrum autofocusing and twin-image suppression algorithms. This allows the estimation of the sample's transmission spectra and realizes pixel-level image registration. The imaging performance of the system is tested using the United States air force (USAF) resolution chart, and multispectral experiments are conducted considering different biological tissue samples and liquids with different properties. The results demonstrate that the system can achieve an imaging resolution of up to 7.81 μm. Furthermore, the proposed imaging technique not only achieves color holographic microscopy imaging with good color reproduction, but also qualitatively distinguish liquids with different properties, which has potential application prospects in the imaging and detection of unlabeled biological samples.