Western blotting is widely used in protein analysis, classically using enhanced chemoluminescence for protein detection. Fluorescence-labelled secondary antibodies have emerged in recent years for detection of antigens, in order to improve the sensitivity and the linear range of detection. Here we show that the sensitivity can be further improved by an additional step in the detection procedure: the antigen is detected by successive incubations with a primary antibody, followed by a biotinylated secondary antibody and then a tertiary fluorescent conjugate. Using the detection of different antigens by CyDye-conjugated secondary antibodies in a two-step protocol as a reference, two tertiary fluorescent conjugates were evaluated: CyDye-conjugated streptavidin and CyDye-conjugated anti-biotin antibody. An four-fold increase in sensitivity was achieved with CyDye-conjugated streptavidin; numerous unspecific bands were also generated. CyDye-conjugated anti-biotin antibody did not generate any unspecific bands and led to a 30-fold increase in sensitivity, compared to detection with CyDye-conjugated secondary antibody.