A fluorescent chitosan derivative was deposited layer-by-layer onto conventional paper sheets and its distribution, in terms of both spreading and penetration, was assessed by SEM observations and emission measurements. The results showed that, on the one hand the surface distribution was highly homogeneous and, on the other hand, the penetration of chitosan within the paper pores ceased after a three layer deposit, beyond which any additional coating only produced an increase in its overall thickness and film-forming aptitude. These results show that this modified chitosan can be used as probe to optimize and understand the mechanism of the deposition of chitosan onto paper and other substrates.