Modulation transfer function (MTF) is one of the important parameters of high-spatial-resolution satellite optical cameras. A new method for in-flight MTF measurement using periodic targets is presented. It can directly obtain the MTF value at Nyquist frequency and MTF plots by parametric model. The experimental results show that the satellite optical camera′s MTF can be directly and accurately acquired by deployment of five groups of non-integer pixel (ground sample distance) interval of three-bar targets in cross and along track directions respectively, and can be arranged with the large-area target in homogenous dark background. The error is less than 5% and the approach can satisfy the application requirements for spaceborne estimation.