The characterization and localization of proteins and other organic components in the complex stratigraphy of paintings is crucial for authentication and studies of painting techniques. With this aim we have developed a new ultrasensitive immunochemical procedure for the detection of the protein ovalbumin (chicken egg white albumin), present in binding media or varnishes, in painting cross-sections. The technique is based on chemiluminescence imaging detection combined with optical microscopy, and allowed the sensitive localization of the target protein in cross-sections with high spatial resolution. In order to evaluate its performance, the method was first applied to standard samples (also containing different common pigments), then used for the localization of ovalbumin in samples obtained from a Renaissance wood painting.