A cross-sectional study was conducted on the three types of working donkeys in Ethiopia (Abyssinian, Ogaden, and Sennar) while they are in their ecological adaptation sites to evaluate and compare the reference values of serum biochemical profiles. Blood samples were collected from a total of 229 apparently healthy adult working donkeys (134 Abyssinian, 55 Ogaden, 40 Sennar types), and ten serum biochemical analytes (total serum protein, glucose, creatinine, gamma glutamyl transferase (GGT), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), sodium, chloride, and potassium) were analyzed using a commercial kit (Centronic GmbH, Wartenberg, Germany) for the target groups. A comparison based on the serum biochemical profile of the three donkey types irrespective of sex has shown significantly lower serum activities of ALT (19.20 ± 3.55 IU/l; CI, 18.06–20.34) and AST (178.13 ± 55.70; confidence interval (CI), 160.31–195.94) for Sennar donkeys compared to the activities of ALT (23.65 ± 7.73; CI, 21.56–25.75) and AST (240.60 ± 110.20; CI, 210.81–270.39) (P < 0.05) for Ogaden-type adult donkeys. A significantly lower serum activity of GGT (36.36 ± 14.70 IU/l; CI, 33.85–38.87) was observed for Abyssinian donkeys than for Ogaden donkeys (48.24 ± 16.59 IU/l; CI, 43.75–52.72) (P < 0.001). The serum sodium (147.55 ± 21.81 mmol/l; CI, 141.65–153.44) and chloride (116.67 ± 16.23 mmol/l; CI, 112.28–121.06) concentrations of Ogaden donkeys were found to be significantly higher than the sodium (132.68 ± 14.16 mmol/l; CI, 128.15–137.20) and chloride (104.50 ± 6.45 mmol/l; CI, 102.44–106.56) (P < 0.001) concentrations of Sennar donkeys. A significantly higher serum potassium concentration (4.84 ± 0.63 mmol/l; CI, 4.73–4.94) of Abyssinian donkeys was found compared to those of Sennar (4.38 ± 0.49 mmol/l; CI, 4.22–4.54) and Ogaden donkeys (4.31 ± 0.78 mmol/l; CI, 4.10–4.52) (P < 0.05). There was no significant variation in the serum concentration of total protein, glucose, creatinine, and ALP activity among the three types of donkeys. A comparison of the serum biochemical profile of adult jacks has shown a significantly different result for the serum activities of GGT, ALT, and AST and the concentrations of serum chloride and sodium among the three donkey types. Abyssinian jennies in Afar had significantly higher serum activities of AST (248.92 ± 120.05 IU/l; CI, 202.78–295.07) (P < 0.05) and ALT (29.04 ± 8.34 IU/l; CI, 25.83–32.24) (P < 0.001) than the AST (192.57 ± 66.60 IU/l; CI, 176.97–208.17) and ALT (20.76 ± 6.61 IU/l; CI, 19.21–22.31) activities for jennies in Sebeta but had lower serum glucose and chloride concentrations. In conclusion, the findings of the present study could be used as reference values for the serum biochemical parameters of the three types of donkeys in Ethiopia, and clinical interpretation of the biochemical parameter based on the standard reference values should consider the types of donkeys.