Amoebiasis is still the cause of one of the major health problem and a common lifethreatening parasitic disease affecting 12% of the world population. The present study was undertaken to evaluate the seroepidemiology of amoebiasis in Najran City, Saudi Arabia. A total of 455 blood samples taken from healthy persons and surplus serum from various hospitals serology laboratories, over a one year period, between September 2014 to September 2015 were screened for the presence of amoebic antibody in their blood serum using indirect hemagglutination assay (IHA). Out of the 455 samples of sera tested, 158 (40.7%) were found to be positive with a titers ranging from 1:32 to 1:512. The younger age groups had the highest t positive rates than the other groups (p<0.0001). The prevalence of infection was more common in females (57.3%) than males (29.8%), and showed statistical significance (P<0.001). There was a significant association on the presence of Amoeba antibodies in relation to the nationality of residence, and the seasonal dynamics of infection (P<0.05). The present study noticed that stray cats are potential reservoirs of infections and should be considered important to public health. The present findings highlighted that Entamoeba histolytica parasite is prevalent in Najran area. The establishment of such data will be beneficial for the public health authorities in the planning and implementation of specific prevention and control strategies of this infection in Saudi Arabia.