The invention discloses a device and a method for the unmarked monitoring of the activity of cells based on lensless diffraction imaging. A host computer controls a collecting hole positioning mechanism to work through a slave computer, drives a lensless diffraction imaging mechanism to move to make the lensless diffraction imaging mechanism align to different culture wells in a 96-well cell culture well plate, and controls a CMOS sensing module to photograph and acquire diffraction images corresponding to all the culture wells, every image is processed to obtain a single-cell diffraction pattern in every image, the pixel intensity contrast and the pattern strip dispersity are calculated, and are compared with preset thresholds to determine whether the cell is a living cell or not, and thecell activity index is obtained through comparing the living cell quantity of a sample group with the living cell quantity of a control group. The device and the method do not need markers, and do not need biochemical reaction operation every once in a while, so the cell activity monitoring accuracy is improved.