The spatial resolution of transmission electron backscatter diffraction (t-EBSD) with a standard conventional EBSD detector was evaluated quantitatively based on the calculation of the correlation coefficient of transmission patterns which were acquired across a twin boundary in the sample of austenitic steel. The results showed that the resolution of t-EBSD improved from tens of nanometres to below 10 nm with increasing accelerating voltage and thinning of specimen thickness. High voltage could enhance the penetration depth and reduce the scattering angle. And the thinning of specimen thickness would result in decreasing of the scattering events according to the theory of thermal diffuse scattering (TDS). In addition, the transmission patterns were found to be weak and noisy if the specimen was too thin, because of the decreasing intensity detected by the screen. Consequently, in this work, the best spatial resolution of 7 nm was achieved at 30 kV and 41 nm thickness. Moreover, the specimen thickness range was also discussed using Monte-Carlo simulation. This approach was helpful to account for the differences of measured spatial resolutions, by t-EBSD, of lamellas with different thickness.