The spherical reference in a vertical Fizeau interferometer was calibrated by the two-sphere method to identify the surface deformations induced by gravity, mounting or holding forces to improve the surface measurement accuracy of optical elements in vertical optical systems. First, the algorithm of two-sphere absolute method was deduced. Then, the factors that may influence measurement accuracy, including environment, gravity, mounting or holding forces were analyzed in theory or calculation by simulation. Finally, the spherical reference used in the vertical Fizeau interferometer was calibrated by two-sphere method, and the experimental results were analyzed by the law of error combination. Experimental results indicate that the calibration accuracy by using two-sphere method is 2.3 nm RMS for the spherical reference of F/15 vertical Fizeau interferometer. Among them, the measurement repeatability induced by the method and experimental operation is no more than 0.7 nm, and that is no more than 1.2 nm while environment influences are taken into account, including vibration and temperature. Furthermore, the surface deformation induced by gravity is about 0.9 nm RMS, and that induced by mounting or holding force is about 1.7 nm. In conclusion, two-sphere method is high precise method that can be used in calibrating vertical spherical reference and the influence of environments on measurement is relative to the length of interferometric cavity. The spherical reference must be calibrated before it is used for high precise measurement vertically, for the surface deformation will be remarkable induced by the gravity, mounting or holding forces.