This study aimed to characterize and determine the prevalence and the associated risk factors of trypanosomiasis in different Egyptian livestock from various governorates using molecular and serological techniques, adding to an assessment of some biochemical alterations in serum samples. A total 1019 blood samples were collected from nine governorates in the period from January 2020 to September 2021 from cattle (n = 752), buffaloes (n = 42), sheep (n = 65), goats (n = 60), donkeys (n = 54), and horses (n = 46). Molecular investigation followed by sequencing confirmed the presence of only Trypanosoma evansi (T. evansi) infection with a prevalence rate of 3.73 % (38/1019). Serologically, T. evansi was detected by indirect ELISA in 11 samples out of 519 negative PCR ones, with an overall prevalence 2.1 %. Furthermore, statistical difference was found between investigated governorates in both molecular and seroprevalence. Surprisingly, the risk of T. evansi infection was significantly (P < 0.05) higher in winter (9.83 %) than in autumn (1.4 %), while no infection was recorded in the spring and summer seasons. No significant difference regarding animal hosts, age, and sex as risk factors was recorded. Serum biochemical analysis showed a non-significant decrease of total proteins, albumin, A/G ratio, and HDL-cholesterol in cattle, equine, and goats with a non-significant increase in total globulins level. In sheep, a significant increase was determined in all tested parameters except a significant decrease in the level of albumin and A/G ratio. To the best of our knowledge, this is the first large-scale epidemiological study determining the endemicity of the neglected T. evansi infection in different animal species in multiple governorates of Egypt. It is recommended to use both molecular and serological techniques for efficient diagnosis of trypanosomiasis as a preliminary approach to control the disease.