Raman spectroscopy is a technique that provides structural information on lignin and other components of wood and pulp in situ. However, especially lignin-containing samples may produce laser-induced fluorescence (LIF) that overlaps with Raman bands. In the worst case, this background signal can overwhelm the weaker Raman signal completely. In this study, the LIF of lignin was investigated with the excitation wavelength 532 nm applied in Raman spectroscopy to clarify the correlations between lignin structure and LIF intensity. Raman spectroscopic analyses with lignin model compounds illustrated that the 5-5′ structures induce LIF. It was also shown that the intensity of LIF was significantly less intense when the 5-5′ model compound was structurally rigid (as in dibenzodioxocin) compared with the flexible simple counterpart. The comparison between the free phenolic model compounds with the methylated analogue showed that the presence of the free phenolic structure was not a prerequisite for LIF. It was thus concluded that the conformation of the molecule is the key factor with respect to fluorescence. The role of conformational aspects was further investigated by comparing wood with chemical pulps and isolated lignins.