Every year new fentanyl analog compounds, or fentanyls, appear on the drug scene. Development of immunoassays dedicated for screening individual molecules is challenging due to the short-lived presence of these compounds on the recreational drug market. Therefore, we investigated the detecting capabilities of the immunalysis fentanyl direct enzyme-linked immunosorbent assay (ELISA) kit against fentanyl in whole blood, and determined the cross-reactivity of nine fentanyl analogs (2-fluorofentanyl, acetylfentanyl, acrylfentanyl, carfentanil, cyclopropylfentanyl, tetrahydrofuranylfentanyl, furanylfentanyl, ocfentanil, valerylfentanyl) to confirm its validity for the general screening of fentanyls. Immunalysis ELISA assay was used to test whole blood samples fortified with fentanyl on a TECAN Freedom EVOlyzer platform, according to manufacturer specifications. The kit successfully was validated for fentanyl screening with a cutoff set at 0.5 ng/mL, and all tested analogs, with the exclusion of carfentanil, were detected. The lowest cross-reactivity with the kit was obtained with furanylfentanyl (20% ± 1, 95% confidence intervals (CI)) and 4-fluoroisobutyrfentanyl (25% ± 1, 95% CI), while the highest was recorded using acetylfentanyl (99% ± 11, 95% CI) and acrylfentanyl (94% ± 10, 95% CI). Post-mortem samples containing fentanyl, acrylfentanyl, cyclopropylfentanyl, THF-fentanyl and 4-fluoroisobutyrfentanyl were screened, and sensitivity and specificity of each analog were calculated. Positive screening results were generated by all post-mortem cases containing fentanyl (n = 14), acrylfentanyl (n = 11), cyclopropylfentanyl (n = 14), tetrahydrofuranylfentanyl (n = 13) and 4-fluoroisobutyrfentanyl (n = 10). Concentration of post-mortem fentanyl samples ranged from 0.5 ng/mL (cutoff) to 230 ng/mL, while the range for analogs was 3.4-36 ng/mL (cyclopentylfentanyl), 0.76-370 ng/mL (4-fluoroisobutyrfentanyl), 0.02-12 ng/mL (acrylfentanyl) and 2-26 ng/mL (tetrahydrofuranylfentanyl). The immunalysis fentanyl direct ELISA kit was successfully validated and showed significant cross-reactivity for all tested fentanyls, except carfentanil, making it a suitable technique for fentanyl and fentanyl analogs screening.