The present study was aimed at designing of immunoenzyme test-system for competitive determination of 25OHD in blood serum using polyclonal antibodies against 25OHD and biotin-streptavidin visualization technique. The validation parameters of test-system were determined. For development of test-system we used the preparation of specific antibodies against 25OHD with the titer 1:3000. The optimal concentration of competing agent (25OHD3-LC-biotin) was found to be equal to 5 ng/ml. The standard calibration curve was built by using a set of seven calibrators. For the validation of test-system the following parameters were established: limit of detection, quantitative limit, cross-reactivity, intra- and inter-system coefficients of variation (Intra.CV, Inter.CV) and “matrix effect”. Constructed immunoenzyme test-system can be used successfully for 25OHD determination in blood serum samples.