The optical characteristics of substrates are the essential content of research work for thin film optics. The
spectrophotometer is one of the important instruments to test transmittance and reflectance of different substrates or
coating components. The test accuracy of transmission spectrum of the substrates has direct influenced on optical
constants reverse engineering of thin film on the substrate. In this paper, SCHOTT grade LITHOSIL_Q0 fused silica
substrate was chosen as standard substrate and was tested transmission spectrum in region 380nm-860nm to use the
Lambda-900 spectrophotometer. By comparing the measured transmission spectrum with standard SCHOTT grade
LITHOSIL_Q0 fused silica substrate transmission spectrum can be calculated measured errors. In the experiments, the
orthogonal experimental design method is used. Sample pool aperture, scan speed, slit width and sampling data interval
operating parameters were chosen as key factors (three levels were chosen in each factor). L9 (34) orthogonal table was
used for analysis the experiments results. Nine experiment results analyzed to determine the operating parameters of the
influence measured accuracy were as follows: the sample pool aperture, scan speed, slit width, the sampling data interval.
Finally, no iris, scan speed 250nm/s, slit width 3nm, sampling data interval 1nm were used a combination of the
operating parameters to measure of transmission spectrum of the SCHOTT grade LITHOSIL_Q0 samples. Measured
maximum and minimum absolute error are 0.1355% and 0.0771%, respectively. The operating parameters could be
applied to transmittance spectrum test of every substrate, or the spectrum could be extended.