UV-LIF measurements were performed on ambient aerosol in Manchester, UK (urban city centre, winter) and Borneo, Malaysia (remote, tropical) using a Wide Issue Bioaerosol Spectrometer, version 3 (WIBS3). These sites are taken to represent environments with minor and significant primary biological aerosol (PBA) influences respectively, and the urban dataset describes the fluorescent background aerosol against which PBA must be identified by researchers using LIF. The ensemble aerosol at both sites was characterised over 2–3 weeks by measuring the fluorescence intensity and optical equivalent diameter (<i>D</i>_P) of single particles sized 0.8 ≤ <i>D</i>_P ≤ 20 μm. Filter samples were also collected for a subset of the Manchester campaign and analysed using energy dispersive X-Ray (EDX) spectroscopy and environmental scanning electron microscopy (ESEM), which revealed mostly non-PBA at <i>D</i> ≤ 1 μm. <br></br> The WIBS3 features three fluorescence channels: the emission following a 280 nm excitation is recorded at 310–400 nm (channel F1) and 400–600 nm (F2), and fluorescence excited at 350 nm is detected at 400–600 nm (F3). In Manchester the primary size mode of fluorescent and non-fluorescent material was present at 0.8–1.2 μm, with a secondary fluorescent mode at 2–4 μm. In Borneo non-fluorescent material peaked at 0.8–1.2 μm and fluorescent at 3–4 μm. Agreement between fluorescent number concentrations in each channel differed at the two sites, with F1 and F3 reporting similar concentrations in Borneo but F3 outnumbering F1 by a factor of 2–3 across the size spectrum in Manchester. <br></br> The fluorescence intensity in each channel generally rose with <i>D</i>_P at both sites with the exception of F1 intensity in Manchester, which peaked at <i>D</i>_P = 4 μm, causing a divergence between F1 and F3 intensity at larger <i>D</i>_P. This divergence and the differing fluorescent particle concentrations demonstrate the additional discrimination provided by the F1 channel in Manchester. The relationships between fluorescence intensities in different pairs of channels were also investigated as a function of <i>D</i>_P. Differences between these metrics were apparent at each site and provide some distinction between the two datasets. Finally, particle selection criteria based on the Borneo dataset were applied to identify a median concentration of 10 "Borneo-like" fluorescent particles per litre in Manchester.