A method of inspecting flatness of substrate is provided and includes providing a substrate. N first inspecting points are selected from the surface of the substrate along a first straight line, where the coordinate of the i-th first inspecting point is (Xi,Yi,Zi). By using a formula “D=Σi=1N−1√{square root over ((Xi+1−Xi)2+(Yi+1−Yi)2+(Zi+1−Zi)2)}”, a first measurement length D is calculated. By using a formula “F=(D−S)/S”, a first flatness index F is calculated. S is the horizontal distance between 1st first inspecting point and N-th first inspecting point. When the first flatness index F is larger than a first threshold, the substrate is determined to be unqualified.