In this work, we report a dual use of highly fluorescent N- and O-doped carbon dots (CDs) for rapid and high-throughput trace analysis of iron in water and organic phases. The CDs are rapidly synthesized in a sealed vessel via microwave irradiation within 5 min, and they exhibit high quantum yields of 80% with sensitive quenching responses to iron contents. Combined with a microplate fluorescence reader, a rapid and high-throughput assay for ions is further developed. The whole process from the CD synthesis to the detection output can be accomplished within 15 min. The limits of detection for Fe3+ in aqueous solution and ferrocene in organic gasoline are determined down to 0.05 mM. Furthermore, this method has been successfully used to determine the level of irons in real gasoline for quality evaluation. The results have an excellent agreement with atomic absorption spectrophotometric measurements. The CD-based facile assay with lower cost, use of less sample, and higher-throughput holds great promise as a powerful tool for iron detection in water and organic phase samples.