Abstract: A spatial omics single cell data acquisition method includes: acquiring a dyeing image of a biological sample, and acquiring a visual gene expression image of the biological sample; registering the dyeing image with the visual gene expression image, so as to obtain a registered image; performing cell segmentation on the registered image, so as to obtain a cell mask image; and according to the cell mask image, determining a cell of a background gene molecule of an unknown cell in the visual gene expression image, and marking the cell, so as to obtain a spatial omics molecular expression matrix with a cell mark. Compared with the prior art, the present disclosure relates to obtaining high-precision spatial single cell data by means of a gene expression matrix diagram, and a processing flow involving splicing, registration, segmentation and correction.