In this paper a new calibration method in diffuse optical tomography (DOT) is presented. A calibration matrix is obtained from a homogeneous phantom measurement together with the forward calculation from a homogeneous medium. Applying this matrix to the measurement data from the heterogeneous medium of interest allows us to remove errors from model mismatch, fibre optic positioning, and numerical noises in inverse computation. It also permits accurate determination of the initial optical properties and boundary conditions coefficient for iterative image reconstruction. A series of two-dimensional (2D) phantom experiments are performed to evaluate the method. The results show that the quality of image reconstruction can be enhanced significantly. In particular, targets with absorption contrast as low as 1.4 can be reconstructed quantitatively. Additional experiments are conducted using varied sizes and optical properties of the reference phantom. It appears that these reference phantom parameters present moderate impact on the final image reconstructions.