Objective To investigate the optimal approach for sample preparation in the process of FISH evaluation.Methods 300 patients diagnosed as pulmonary tuberculosis and 300 subjects with ruled-out pulmonary tuberculosis were enrolled and served as positive and negative groups respectively.800 suspected patients of pulmonary tuberculosis were also enrolled as research group and underwent sputum smear,culture for mycobacterium tuberculosis,real-time PCR and FISH evaluation.Two approaches of paraffin embedding for pulmonary tissues were used.The first one was self-created novel method and the second one was as traditional process.The sensitivity and specificity of these two methods were compared.Results (1) The positive rate of the first approach was 98.7％ and the second approach was 97.9％ and there was no statistical significant difference between these two methods (P=0.148).(2) The negative rate of the first approach was 97.8％ and the second was 96.1％ and there was statistical significant difference between these two methods (P=0.036).Conclusion FISH was valuable for the tuberculosis detection and optimizing the process would increase the sensitivity and specificity of FISH evaluation.