This study aimed to evaluate the efficiency of single and continuous testosterone injection against cockscomb size, T3/T4 ratio, and histopathological features of the bursa of Fabricius in the male layers. Sixty DOC layers were divided into 3 groups namely control (C), single testosterone injection with a dose of 3 mg/day (TO), and continues testosterone injection induced for 35 days (TC). One mL of blood was collected through the brachial vein, cockscomb measurements were performed serially every week for 5 weeks, while the bursa of Fabricius organ was carried out subsequently after the animal was sacrificed. Analysis of thyroid hormones was performed using ELISA, while the histopathological features were performed by HE staining. The results showed that cockscomb size was C: (6.29±1.62), TO: (7.79±0.21), and TC: (8.64±1.58) cm 2 . However, there was no difference between TO and TC at week 5 (P > 0.05). There was a significant decrease of the T3/T4 ratio at week 3, control: 3.24±0.18; TO: 3.76±0.39; and TC: 2.44±0.14. At the age of 35 days, crystals had been formed in the bursa of Fabricius in group TO and TC. In conclusion, testosterone injection affects cockscomb size, T3/T4 ratio, and accelerates degeneration of the bursa of Fabricius. Furthermore, the administration of a single dose of testosterone injection is more efficient than the continuous one.