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The work was carried out to develop an immunoassay for estimation of Aspergillus ochraceus biomass on solid substrate.An indirect noncompetitive enzyme-linked immunosorbent assay (ELISA) was developed for determination of fungal biomass in food commodities using antibody raised against A. ochraceus mycelial antigen. The sensitivity of the assay was linear in the range of 10–160 μg fungal biomass per millilitre extract of coffee (R2 = 0·989), poultry feed (R2 = 0·987) and chilli (R2 = 0·989). The growth of A. ochraceus in the food commodities like chilli, coffee beans and poultry feed, under the influence of two levels of moisture (20% and 30%) were monitored by the ELISA. The maximum fungal colonization was observed in poultry feed (9·8 and 11·8 mg g−1) followed by coffee beans (6·8 and 11·3 mg g−1) and chilli (5·1 and 6·3 mg g−1) at 20% and 30% moisture after 20 days of incubation. Similarly the fungus produced maximum ochratoxin A in poultry feed (25 and 120 μg g−1) followed by coffee beans (8 and 24 μg g−1) and chilli (0·2 and 0·45 μg g−1) at 20% and 30% moisture after 20 days of incubation.The method can be used for quantitative estimation of fungal biomass and comparison of fungal colonization in food substrates varying in composition.The method can be adapted for studying the fungal colonization in different solid substrates under different culture condition. The method is sensitive to mould colonization of ≥0·02% (w/w) and can be used for early detection of specific fungal infestation in food commodities.