ObjectiveTo evaluate the utility of a proposed cell transfer technique for constructing cytological smear microarrays and its potential applications in multiplex immunocytochemical (ICC) staining.MethodsNinety‐six cytology smears, including two pericardial effusions, 22 ascites and 72 pleural effusions, were transferred to a 33‐plex cytological microarray. Paired staining of thyroid transcription factor‐1 (TTF‐1) and calretinin ICC was performed in duplicate slides.ResultsMost of the smeared cells selected for transfer could be removed from the original slides with a minimal loss of cells and with no change in morphological features or immunoreactivity. Comparison of the staining results with immunohistochemical staining results, clinical history and histopathological reports available for each patient revealed that TTF‐1 was positive in 32/33 metastatic pulmonary adenocarcinomas (PACs), 1/15 non‐pulmonary adenocarcinomas and 0/45 benign effusions. The ICC results for TTF‐1 on a transferred cytological microarray revealed high (97%) sensitivity and high (96.7%) specificity for the detection of metastatic PAC.ConclusionCytology microarrays can be constructed by transferring cells from serous fluid cytological smears, and cells transferred to the microarray retain their morphological integrity and immunoreactivity. Researchers can use the technique for simultaneous immunostaining of multiple specimens in studies of neoplastic or non‐neoplastic diseases when available tissue samples are limited.