In this paper, a new method for retrieving of molecular oxygen column density of the middle and upper atmosphere is presented from nadir measured data. From the cross section of absorption for oxygen molecules, we can know that the absorption of molecular oxygen in N2 Lyman-Birge-Hopfield short (LBHS) is strong but it is weak in N2 Lyman-Birge-Hopfield long (LBHL). Based on this characteristic, we propose that the nadir radiation intensity ratio of LBHS to LBHL, ln(LBHS/LBHL) can be used to retrieve molecular oxygen column density of the middle and upper atmosphere. An interpolation table for estimating the molecular oxygen column density is calculated from the airglow model. This table includes ln(LBHS/LBHL) for different oxygen contents, different solar zenith angles, satellite observation angles and solar activity conditions. Then, we get the molecular oxygen column density from ln(LBHS/LBHL) and the corresponding solar zenith angle, satellite observation angle and solar activity index. Finally, we use mode simulation to evaluate the inversion result, which proves that this new method can successfully obtain molecular oxygen column density of the middle and upper atmosphere.