In this paper, we report a precision measurement of hyperfine splitting and absolute frequency of D1 line in cold 6Li atoms. The gray molasses is realized in the experiment and the tempreature is cooled to about 50 μK, which is lower than the Doppler cooling limit, 140 μK. By use of an optical comb, the absolute frequencies and corresponding hyperfine splitting are measured. We obtain frequencies of 446789503.080(35) MHz, 446789529.198(36) MHz, 446789731.316(50) MHz and 446789757.476(29) MHz for the D1 line. The results are in reasonable agreement with the theoretical calculations and consistent with earlier measurements. They could provide an important foundation for future frequency measurement, α constant and nuclear radius.