To diagnose the trematode Cotylophoron cotylophorum by the sedimentation and sieve technique has low efficiency when the parasite burden is low, because such technique has low sensitivity to diagnose the trematode eggs in the fecal samples. The main objective of this research was to purify some proteins from the secretory and excretory products of adult stages of C. cotylophorum with the aim of assessing its antigenicity by immunoelectrotransferency (Western Blot technique), next those proteins were used as antigens in an enzyme link immune assay (ELISA). First, 1,200 adult C. cotylophorum, collected directly from bovine´s rumen slaughtered at the local Matadero Industrial Centroccidental (MINCO), were incubated during 16 hours in Minimun Essential Eagle (MEM), and antigenic proteins were obtained from this MEM. Next, proteins were purified and concentrated by ultracentrifugation. An electrophoresis technique (SDS-PAGE) and a Western Blot were carried out to identify the antigenic proteins using an hyperimmune serum obtained from previously immunized rabbits with the purified proteins isolated from the excretory and secretory products of C. cotylophorum. Nine (9) protein bands were identified with molecular weights of: 17, 24, 43, 56, 62, 76, 83, 105, and 121 KDa, and three of these bands (62, 76, and 105 KDa) were recognized by the serum from four (4) bovines which had previously been diagnosed as positives to C. cotylophorum by coprologic tests. The protein with 76 KDa was the most reactive. Finally, these purified antigens may be used to develop immunoenzymatic assays with greater sensitivity and specificity, which would be very helpful tests for the diagnostic and epidemiologic study of C. cotylophorum in Venezuela.El diagnóstico de Cotylophoron cotylophorum por la técnica sedimentación y tamizado, es poco eficiente cuando la carga parasitaria es baja y tienen un poca sensibilidad; al diagnosticar sólo la presencia de huevos en heces. En el presente estudio se purificaron proteínas del producto de excreción – secreción de formas adultas de C. cotylophorum para evaluar su antigenicidad en una inmunoelectrotransferencia (Western Blot) y su uso diagnóstico en un inmunoensayo (ELISA). Los antígenos obtenidos después de 16 horas de incubación en medio Mínimum Essential Eagle (MEM), de 1200 especímenes de parásitos adultos de C. cotylophorum</i>, recolectados del rumen de bovinos sacrificados en el Matadero Industrial Centroccidental (MINCO), fueron purificados y concentrados por ultra filtración. Las proteínas antigénicas se identificaron en una electroforesis (SDS – PAGE) y Western Blot utilizando suero hiperinmune obtenidos de conejas inmunizadas con las proteínas purificadas del producto de excreción y secreción de C. cotylophorum. Nueve bandas fueron identificadas con pesos moleculares de: 17, 24, 43, 56, 62, 76, 83, 105 y 121 KDa, y tres de estas bandas (62, 76 y 105 KDa), fueron reconocidas por el suero sanguíneo de 4 bovinos positivos por examen coprológico a C. cotylophorum, de las cuales la proteína de 76 kDa mostró la mayor reactividad. Los antígenos purificados pueden ser usados para desarrollar ensayos inmunoenzimáticos más sensible y específico, de gran utilidad en el diagnóstico y estudio epidemiológico de C. cotylophorum en Venezuela.