Objective To explore the application value of double culture method in improving the detection rate of smear-negative Mycobacterium. Methods The screened specimens of patients with the initial diagnosis in a hospital from January 2019 to September 2021 were collected and grouped according to solid culture, liquid culture and solid-liquid double culture. In addition, patients with smear-negative Mycobacterium, positive solid, positive liquid or positive solid-liquid double culture were selected, they were divided into pulmonary Mycobacterium (PM) group and extrapulmonary Mycobacterium (EPM) group according to specimen sites, clinical characteristics of two groups of patients were analyzed. Results A total of 6 492 smear-negative patients were surveyed, 3 234 were in solid group, 1 938 in liquid group and 1 320 in double culture group, 253 patients (3.90%) were positive for culture, positive culture detection rates of patients in solid culture, liquid culture and solid-liquid double culture group were 2.35% (n=76), 4.54% (n=88) and 6.74% (n=89) respectively; detection rates of PM were 3.11%, 6.02% and 7.13% respectively, detection rates of EPM were 1.02%, 2.68% and 6.09% respectively. Among 253 smear-negative but culture positive patients, 143 were male and 110 were female; the mean age was (58.34±18.47) years; the median detection time were 34.65, 10.45 and 9.89 days respectively. Among patients in PM group, the proportion of male patients, cough and expectoration, underlying respiratory diseases, inflammatory exudation showed on chest CT, positive results of tuberculosis T cell spot test were all higher than EPM group, differences were all significant (all P < 0.05). Positive detection rate of EPM of solid-liquid double culture group was higher than solid and liquid groups (both P < 0.001), positive detection rate of PM in solid-liquid double culture group was higher than solid culture group (P < 0.001). Conclusion Patients with PM have the characteristics of cough and expectoration, respiratory diseases, inflammatory exudation on chest CT, while patients with EPM have no typical characteristics. Clinical signs of EPM are more concealed than that of PM, and it is easy to miss diagnosis, solid-liquid double culture method can improve the detection rate of Mycobacterium in optimizing the sample pre-treatment and workflow.