Photoluminescence (PL) multiplexing usually relies on spectral or temporal separation. A combination into higher-order multiplexing for biosensing is extremely challenging because the PL intensity is required for target quantification at very low concentrations and the interplay of color, lifetime, and intensity must be carefully adapted. Here, we demonstrate time-gated Förster resonance energy transfer (TG-FRET) from a long-lifetime Tb complex to Cy3.5 and Cy5.5 dyes for spectrotemporal multiplexing of four different DNA targets in the same sample by single-color excitation and two-color detection. We used rolling circle amplification (RCA) for high specificity and sensitivity and for placing Tb donors and dye acceptors at controlled distances within the amplified DNA concatemers. This precise distance tuning led to target-specific PL decays of the FRET pairs and simple, separation-free, and higher-order multiplexed quantification of DNA. The RCA-FRET DNA assay could distinguish very homologous target sequences and provided limits of detection down to 40 zeptomoles (300 aM).