The current study compared the performance of serum and milk ELISA in determining Neospora caninum prevalence in lactating Nili-Ravi buffaloes. Overall, 64 milk samples (n=57 individual milk samples; n=7 bulk milk samples) collected from buffaloes of districts Lahore and Narowal were tested for anti-N. caninum antibodies using milk iscom ELISA. The same animals were evaluated serologically using serum cELISA for this purpose. Furthermore, hematological profiling of 50 seropositive and 15 seronegative buffaloes was performed to appraise any effect of N. caninum seropositivity on the hematological parameters of the host. Resultantly, milk ELISA detected lesser milk samples positive i.e., 61.64% (±12.6, 95% C.I. 35/57) for N. caninum against higher seropositivity (76.6% ±7.16, 42/57) by serum cELISA. The agreement level (Kappa ratio=0.568 & R 2 =0.769) between the performance of two assays was found good. General Linear Model analysis of hematological parameters reflected significantly (P<0.05) lower monocyte count but higher blood glucose levels in seropositive buffaloes associated with different seasons. It was concluded that, owing to the limitations of the milk ELISA (also lower sensitivity than cELISA) it is best suited for finding prevalence in lactating dairy herds having substantial number of infected animals but in general, serum ELISA is best for prevalence studies. While, decreased monocyte count and higher blood glucose levels in the affected animals was observed, which may be attributed to persistent inherent stress of infection on the host.To Cite This Article: Nasir A, Zaman MA, Shakoor A, Rehman TU, Khan I, Ehtisham-ul-Haque S, Sikandar A, Masood S and Akbar A, 2018. Prevalence of Neospora caninum using milk and serum ELISA and its hematological effect in dairy buffaloes. Pak Vet J, 38(3): 281-285. http://dx.