A non-invasive detection method for the status analysis of cell culture is presented based on digital holography technology. Lensless Fourier transform digital holography (LFTDH) configuration is developed for living cell imaging without prestaining. Complex amplitude information is reconstructed by a single inverse fast Fourier transform, and the phase aberration is corrected through the two-step phase subtraction method. The image segmentation is then applied to the automatic evaluation of confluency. Finally, the cervical cancer cell TZMbl is employed for experimental validation, and the results demonstrate that LFTDH imaging with the corresponding image post-processing can provide an automatic and non-invasive approach for monitoring living cell culture.