This study was undertaken with the aim of determining reference hematological and serobiochemical values for working Ethiopian donkeys in four districts of Oromia regional state. The study was conducted by taking blood and serum samples from a total of 130 apparently healthy donkeys which were analyzed by using automated hematology analyzer and photometer 5010, respectively. There were significant difference in RBC (p<0.001), PCV (p<0.04), MCV (p<0.001) and MCHC (p<0.001) among the different age groups. The differences in mean values for PCV, Hb, MCV, MCHC, MCH, Hb, total WBC, eosinophils, monocytes and Platelets between sexes were not statistically significant (p>0.05). In the serobiochemical studies significant difference was seen only in the mean creatinine (p<0.003) values among the different age groups. Therefore, the variations in hematological and serum biochemical values ascribed to the effects of age and sex should be taken into consideration in interpreting physiological hematological and serum biochemical parameters in working donkeys.