Penicillium chrysogenum clade 4, is a common mold on damp building materials. A capture ELISA assay for the major allergen from P. chrysogenum Pch52 has been developed and tested in house dust samples and potential cross-reactivity examined. Minimal cross-reactivity with other relevant indoor fungi was observed for the assay following thorough purification of the monoclonal and polyclonal antibodies. The limit of quantification for ELISA analysis of Pch52 in sieved house dust is comparable to other assays of other fungi. The LOQ for Pch52 was 0.31 ng/mL in solution or 110 ng/g dust. The LOQ for Asp f1 and Alt a1 were 2.2 ng/g and 17 ng/g, respectively. These results indicate this assay is suitable for the quantification of Pch52 in sieved house dust.