In Ti-6Al-4V titanium alloy micro-machining, since the uncut chip thickness (UCT) is comparable to the radius of the tool cutting edge, there exists a minimum uncut chip thickness (MUCT), and when the UCT is smaller than the MUCT, the plowing effect dominates the cutting process, which seriously affects the machined surface quality and tool life. Therefore, the reliable prediction of the MUCT is of great significance. This paper used Deform to establish an orthogonal cutting simulation model, studied the effect of the dead metal zone (DMZ) on the micro-cutting material flow, determined the DMZ range, and proposed a new method for determining the MUCT based on the DMZ. Cutting experiments were conducted to verify the accuracy of the simulation model firstly by cutting force, and then confirming the accuracy of the DMZ-based MUCT determination method through chip analysis and surface quality analysis. Finally, the effects of different cutting conditions on DMZ and MUCT were further investigated using the proposed DMZ-based MUCT determination method. The results show that the MUCT of Ti-6Al-4V titanium alloy is 4.833 μm for a tool cutting edge radius of 40μm and a cutting speed of 10 m/min. The DMZ boundary can be used as the boundary of micro-cutting plastic deformation, and the ratio of MUCT to cutting edge radius, hp/rn will gradually decrease with the increase in the tool cutting edge radius and the cutting speed.