The “turn-off” mode lateral flow immunoassay (LFA) based on the conventional competitive format and “turn-on” mode LFA mainly determined by inner filter effect (IFE-LFA) were objectively compared using T-2 toxin as a model molecule and T-2-BSA and anti-T-2 monoclonal antibody as immunocomplex pairs to improve the sensitivity of the LFA. First, Au nanoparticles (Au NPs), amorphous carbon nanoparticles (ACNPs), quantum dots nanospheres (QDs), and time-resolved fluorescent microspheres (TRFMs) were selected as labels for developing the “turn-off” mode LFAs (Au NPs-LFA, ACNPs-LFA, QDs-LFA, and TRFMs-LFA, respectively). Thereafter, Au NPs and ACNPs were used as absorbers, and QDs and TRFMs were selected as fluorescers, for preparing the “turn-on” mode IFE-LFAs (IFE-LFA based on Au NPs as absorbers and QDs as fluorescers Au NPs-mAb-QDs-BSA, IFE-LFA based on Au NPs as absorbers and TRFMs as fluorescers Au NPs-mAb-TRFMs-BSA, IFE-LFA based on ACNPs as absorbers and QDs as fluorescers ACNPs-mAb-QDs-BSA, and IFE-LFA based on ACNPs as absorbers and TRFMs as fluorescers ACNPs-mAb-TRFMs-BSA). Under optimized conditions, the naked-eye observation cut-off values for the aforementioned eight LFAs were 4, 2, 2, 2, 3, 2, 1.5, and 1 ng/mL, with quantitative limit of detection values of 0.50, 0.23, 0.24, 0.23, 0.47, 0.45, 0.27, and 0.22 ng/mL, respectively. Among all LFAs, ACNPs-mAb-TRFMs-BSA showed the highest sensitivity. The results of this study provide a perspective for improving the sensitivity of the LFAs to meet the requirements of on-site screening for trace substances. Display omitted •“Turn-off” and “turn-on” mode LFAs using the same immune reagent and target were objectively compared.•Dual readout detection and lower immune reagent consumption are significant characteristics of “turn-on” mode LFA.•ACNPs with super absorption performance (250–700 nm) as fluorescence quencher are more conducive to improve “turn-on” mode LFA’s sensitivity.•The capacity of reducing matrix effects by TRFMs as fluorescence donors in “turn-on” mode LFA was better than that of QDs.