A novel nanoknife with a buffering beam is proposed for single-cell cutting. The nanoknife was fabricated from a commercial atomic force microscopy (AFM) cantilever by focused-ion-beam (FIB) etching technique. The material identification of the nanoknife was determined using the energy dispersion spectrometry (EDS) method. It demonstrated that the gallium ion pollution of the nanoknife can be ignored during the etching processes. The buffering beam was used to measure the cutting force based on its deformation. The spring constant of the beam was calibrated based on a referenced cantilever by using a nanomanipulation approach. The tip of the nanoknife was designed with a small edge angle 5° to reduce the compression to the cell during the cutting procedure. For comparison, two other nanoknives with different edge angles, i.e. 25° and 45°, were also prepared. An in situ single-cell cutting experiment was performed using these three nanoknives inside an environmental scanning electron microscope (ESEM). The cutting force and the sample slice angle for each nanoknife were evaluated. It showed the compression to the cell can be reduced when using the nanoknife with a small edge angle 5°. Consequently, the nanoknife was capable for in situ single-cell cutting tasks.