Evaluating renal function is essential for managing captive wild animals, particularly threatened species like the Amazonian manatee (Trichechus inunguis) in rehabilitation and prerelease programs. A series of urine diagnostic tests, such as gross appearance, semiquantitative chemical analyses, microscopic review of sediments, and quantitative analyses of urea and creatinine, were performed in 57 free-catch urine samples. On the same occasion, 52 serum samples from the same individuals were analyzed for creatine kinase activity, creatinine, blood urea nitrogen, albumin, sodium, potassium, and chloride concentrations; serum symmetric dimethylarginine (SDMA) was measured for the first time in the species. Females had higher urine creatinine concentration (21.7 ± 11.8 mg/dl) and specific gravity (1.003 ± 0.003 mg/dl) than males (11.9 ± 8.5 mg/dl and 1.001 ± 0.002 mg/dl) (P < 0.01 for both). Males presented higher urine creatinine:urea ratio than females (24.8 ± 29.2 and 20.5 ± 29.2, respectively) (P < 0.05). Urine pH was lower in manatees from the semicaptive area (7.8 ± 0.6) than in manatees under human care (8.5 ± 0.6) (P = 0.02) using the benchtop pH meter but showed no difference between areas in the dipstick reaction (P = 0.71), given its lower sensitivity and narrower range detection. Furthermore, 18 and 75% of urine samples were positive for the presence of blood and/or nitrite, respectively, in the dipstick reaction, possibly because of sample contamination. Significant correlation was observed between serum blood urea nitrogen and urine urea concentrations (P = 0.02), and between serum and urine creatinine concentrations (P = 0.01). Serum SDMA did not differ between sex or between captive and semicaptive individuals. However, it positively correlated with urine (P = 0.02) and serum creatinine (P = 0.01) demonstrating its potential as a bioindicator of kidney function. The renal function of Amazonian manatees can be effectively assessed by urinalysis, especially when combined with serum biochemistry.