SummaryWe present a numerical analysis and experimental characterisation of spatial resolution in optical projection tomography (OPT) and light‐sheet fluorescence microscopy (LSFM) using their ‘standard’ systems. Although both techniques provide spatial resolution at the micrometre scale for mesoscopic (millimetre to centimetre) samples, LSFM provides higher lateral (∼3 μm, ∼34% of OPT) but lower axial (∼25.8 μm, 295% of OPT) resolution as compared to OPT (∼8.75 μm, 100%) when imaging the same sample (∼2 mm). Moreover, OPT provides isotropic spatial resolution due to its rotational scanning which may reduce the ambiguity in 3D analysis, so it is more practically appropriate for relatively large samples. We also demonstrate the application performances of both techniques by imaging various biological tissues, illustrating their imaging ability at different spatial scales.Lay DescriptionOptical projection tomography (OPT) and light‐sheet fluorescence microscopy (LSFM) are generally used to extract 3D information from relatively large biological tissues/organs/embryos or even some small animals. Both techniques have made a great progress in recent decades and have been widely applied in life science, medical research and so on. The different implementation features of these two techniques results in isotropic and anisotropic spatial resolution respectively, making a dilemma for the researchers to choose the appropriate system when imaging the samples with different size. So far, there is no study to numerically discuss the differences between their image formation properties and to adequately quantify their own strengths and limitations.In our work, we quantified the imaging behaviour in ‘standard’ OPT and LSFM using both numerical analysis and experimental characterisations, showing the relationship between spatial resolution and sample size in each system. We also demonstrated the detailed structure differences when imaging various biological tissues. We believe this work will be useful and can provide a reference for the 3D fluorescence‐imaging‐based researchers.