Preservation of ancient paintings has attracted much attention of researchers for a long time. In the past few decades, many kinds of synthetic polymers and traditional materials had been applied to deal with the degradation of these precious heritages and get a lot of achievements. Through the accumulation of these works, more and more researchers realized the importance of analyzing the composition of painting materials. However, the organic materials in these paintings were not studied in detail at that time due to the limit of precise analytical technology or the immaturity historic preservation concept. Under the situation of consolidants, the high sensitivity and specificity of immunological techniques were still effective or not? This study is aimed at investigating the occurrence of possible analytical interferences from the polymer commonly present in artworks as consolidants and the gelatin solution. Enzyme-linked immunosorbent assay (ELISA) as a highly specific analytical tool for unambiguous recognition has been used in this study and successfully applied to three micro-samples from historical paintings. Our results suggest that the ELISA assay is still useful in the presence of synthetic polymer consolidants, although they reduce the ELISA signal; the gelatin solution would affect the protein identification causing analytical interferences.