Objective: To study the clinical expression significance of seminal plasma biochemical indicators and sex hormone levels in patients with azoospermia and different pathological types through ultrasound examination. Methods: 80 cases of azoospermia in our hospital were selected as experimental subjects, and the cases were collected from January 2021 to December 2024, and according to the pathological typing results of testicular biopsy, all the subjects were divided into 25 cases of hypospermia group, 12 cases of spermatogenic dysfunction group, and 43 cases of supportive cell syndrome (SCOS) group. All underwent ultrasound examination and testicular biopsy. Seminal plasma biochemical indexes [fructose, elastase, neutral alpha-glucosidase (NAG)] and sex hormone levels [follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone (T)] were detected in all three groups, and the results were compared among the three groups. Results: There was no significant difference in the comparison of elastase levels among the three groups (P > 0.05); fructose levels were higher in the hypospadias and SCOS groups compared with the spermatogenic blockage group, and fructose levels were higher in the SCOS group compared with the hypospadias group; NAG levels were higher in the hypospadias and SCOS groups compared with the hypospadias group, and compared with the spermatogenic blockage group, SCOS group NAG levels were higher (P < 0.05); FSH and LH levels were higher and T levels were lower in the spermatogenic dysfunction group and SCOS group compared with the hypospadias group; FSH and LH levels were higher and T levels were lower in the SCOS group compared with the spermatogenic dysfunction group (P < 0.05). Compared with the hypogonadotropic hypogonadism group, both the hypogonadotropic hypogonadism group and the SCOS group exhibited smaller left and right testicular volumes. When compared with the hypogonadotropic hypogonadism group, the SCOS group demonstrated smaller left and right testicular volumes (P < 0.05).Conclusion: There are obvious differences in seminal plasma biochemical indexes such as NAG, fructose and other sex hormone levels such as FSH and LH in patients with different pathological types of azoospermia, and clinics can actively detect the levels of the above indexes so as to assist in determining the degree of testicular function damage and pathological types of patients, and to guide the clinics to carry out targeted treatment.