Abstract: In order to explore the effects of tetracycline hydrochloride (TCH) and alizarin red S (ARS) on Spinibarbus sinensis and their marker effects, and to enrich the fluorescent marking patterns of fish, TCH (from 100 to 500 mg/L) and ARS (from 100 to 300 mg/L) were used for double immersion marking juvenile Spinibarbus sinensis with 6 experimental groups for 24h. The results indicated that 24h double immersion produced detectable double marks in otoliths (sagittae and asteriscus), barbs, fin rays, and fin spines after 90 days in the laboratory growth experiment, with the exception of parts of scales. Yellow fluorescent rings produced by TCH were closer to the inside of the bony structures than red fluorescent rings produced by ARS. Sagittae (≥300 mg/L TCH and ≥150 mg/L ARS), asteriscus (≥300 mg/L TCH and ≥200 mg/L ARS), and barbs (400—500 mg/L TCH and 150—300 mg/L ARS) showed acceptable fluorescent marks at higher concentrations (n≥2). The fluorescence marking of lateral scales and non-lateral scales were not obvious in all treatment groups (0≤n≤1). Fin rays treated with 200—500 mg/L TCH and 150—300 mg/L ARS and fin spines treated with 300—500 mg/L TCH and 200—300 mg/L ARS simultaneously had both acceptable TCH and ARS marks (n≥2). In addition, double fluorescence labeling did not impact fish survival or growth (P>0.05) in the present study. The results suggest that double immersion with TCH and ARS is suitable for double mass-marking juvenile S. sinensis, and these double marks are useful in the experimental development of biological research or restocking methodologies.