The tomato leafminer Tuta absoluta is an important insect pest of tomato crops worldwide. Biological control using entomopathogenic viruses such as isolates of Phthorimaea operculella granulovirus (PhopGV) can be a promising tool for suitably controlling this pest. However to date, few viral isolates from T. absoluta have been reported. Therefore, the aim of this study was to develop an economic, fast and accurate immunological technique for granulovirus detection and to evaluate viral presence in T. absoluta larvae from field samples collected in Colombia. Polyclonal antibodies against PhopGV were produced by using hens and rabbits. The production system in hen was selected as the most efficient and simple method. Then, a dot-blot test was developed, demonstrating the ability of antibodies to recognize granulovirus. Finally, granulovirus presence in 1186 T. absoluta larvae collected in the most important tomato production areas in Colombia was evaluated, and eight isolates were found in Boyaca and Cundinamarca (0.7% of occurrence). These results constitute the first report of a granulovirus isolated from T. absoluta larvae which had been naturally infected in Colombia's tomato producing-fields, and thereby validates the use of the immunological technique to detect granulovirus in field conditions.