Transect lines were laid down in Mkomazi National Park for the wild animals ground count exercise. This technique is applied as a ground truth of population density of animals. Forty transects were set to represent all major habitats, including grasslands, woodlands, forests, shrub land, scrubland, and riverine. Program DISTANCE was employed for data analysis. Normal cosine function was applied without any truncation from which mean population densities of each species were calculated separately with standard errors, encounter rate, and detection probability. A total of 22 species mean population densities were estimated, whereby African buffalo (Syncerus caffer) was leading, while gerenuk (Litocranius walleri) was the least. The Park is well protected at the core zone; however, areas close to boundaries are facing frequent disturbances from humans. Since some species of animals such as large carnivores and elephants were not observed, camera traps should be used to estimate carnivore population, whereas dung counts can be used to estimate elephant density. Continual investment in anti-poaching operations by managers is important to keep safeguarding habitats for wildlife. We recommend the use of these results as a benchmark for future population estimates, and these 40 transects be regarded as standard in future counting.