To investigate the serum levels of reproductive hormones in men with azoospermia and explore the correlation between abnormal reproductive hormones and the results of testicular biopsy and seminiferous extraction. The serum levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (T), and pituitary prolactin (PRL), were measured by Radio immunoassay. Testicular biopsy was used to determine whether patient's had spermatozoa. Spearman's correlation analysis was used to analyse the correlation between LH, FSH, testicular volume, and spermatogenic function. Receiver operator characteristic (ROC) curves were used to evaluate the efficacy and significance of LH and FSH for the detection of spermatozoa and copy number variation sequencing (CNV‐seq). Compared with the control group, the serum levels of FSH and LH in the azoospermia group were significantly up‐regulated (p < 0.001). Prolactin (PRL) and testosterone levels did not differ significantly between the two groups (p > 0.05). The serum levels of FSH and LH were negatively correlated with testicular spermatogenic function and testicular volume. The areas under the ROC curves for determining FSH and LH potency by testicular biopsy with or without spermatozoa were 0.640 and 0.622, respectively. The areas under the ROC curves for the potency of FSH and LH, as judged by CNV detection results, were 0.523 and 0.534, respectively. Serum levels of FSH and LH are associated with azoospermia and may be of predictive significance in the clinical setting for the acquisition of spermatozoa by testicular biopsy.