The optical properties of sulforhodamine B (SRB) hosted in surfactant-modified layered double hydroxide (LDH) are explored using quantum yield efficiencies and temporal variations in luminescence. The effect of the spacer, known to provide a suitable SRB dilution in the solid state, is studied in-depth with respect to its relative content and its alkyl chain length. Counter-intuitively, considering the nature of such a “passive” spacer, the study reports the effect on the luminescence of the optically active molecules for a large series of surfactants. Spacer intercalation enables the distance between adjacent LDH sheets to be adjusted to the size of the luminescent molecule guest, as exemplified here using a series of surfactant molecules: (CH3)(CH2)nSO4Na with odd values 7 ≤ n ≤ 17 and a fixed amount of SRB at 0.05% of the total anion content in the surfactant and SRB. The effect of the surfactant on the optical properties of SRB is investigated, and the best performance is obtained for n = 11, corresponding to the powder LDH–DS/SRB-5. A computational approach evaluating the packing and structural arrangement present in the series of interleaved surfactants helps to interpret the effect of the length of the alkyl chain on the distribution of the SRB within the LDH interlayer. Silicon composite films were elaborated with different LDH loadings, and the composite film with a loading of 40 wt% of LDH–DS/SRB-5 exhibited the maximum absolute photo-luminescence quantum yield (PL QYabs), as high as 60.0 ± 3.0%, being recorded at 570 nm. Furthermore, an in-depth study of fluorescence lifetimes enables us to highlight a rarely-observed phenomenon of delayed fluorescence, and to distinguish for the first time between the location of SRB, either on or inside the LDH platelets.