Abstract Background Hematological studies of any animal species comprise an important diagnostic method in veterinary medicine and an essential tool for the conservation of species. In Sri Lanka, this essential technique has been ignored in studies of many species including reptiles. The aim of the present work was to establish a reference range of hematological values and morphological characterization of wild spectacled cobras (Naja naja) in Sri Lanka in order to provide a diagnostic tool in the assessment of health condition in reptiles and to diagnose diseases in wild populations. Methods Blood samples were collected from the ventral caudal vein of 30 wild-caught Naja naja (18 males and 12 females). Hematological analyses were performed using manual standard methods. Results Several hematological parameters were examined and their mean values were: red blood cell count 0.581 ± 0.035 × 106/μL in males; 0.4950 ± 0.0408 × 106/μL in females; white blood cell count 12.45 ± 1.32 × 103/μL in males; 11.98 ± 1.62 × 103/μL in females; PCV (%) in males was 30.11 ± 1.93 and in females was 23.41 ± 1.67; hemoglobin (g/dL) was 7.6 ± 0.89 in males and 6.62 ± 1.49 in females; plasma protein (g/dL) was 5.11 ± 0.75 in males and 3.25 ± 0.74 in females; whereas cholesterol (mg/mL) was 4.09 ± 0.12 in males and 3.78 ± 0.42 in females. There were no significant differences in hematological parameters between the two genders except for erythrocyte count, thrombocyte count, hematocrit, hemoglobin, plasma protein, percentage of azurophil and heterophil. Intracellular parasites were not found in any of the studied specimens. Conclusion Hematological and plasma biochemical parameters indicated a difference between geographically isolated populations and some values were significantly different between the two genders. These hematological results provide a reference range for Sri Lankan population of adult Naja naja.