High interference and narrow application range are key of bottleneck of recent fluorescence analysis methods, which limit their wide application in the sensing field. Therefore, to overcome these disadvantages, a ratiometric fluorescence sensing system utilizing berberine (BER) and silver nanoclusters protected by dihydrolipoic acid (DHLA-AgNCs) was constructed for the first time in this work, to achieve determination of BER and daunorubicin (Dau). BER aqueous solution (non-planar conformation) has no fluorescence emission. When it was mixed with DHLA-AgNCs, the conformation of BER became planar, producing fluorescence emission at 515 nm besides the fluorescence emission peak of DHLA-AgNCs at 653 nm. With the increase of BER concentration added in system, the fluorescence intensity of BER (planar conformation) at 515 nm increased obviously and the fluorescence intensity of DHLA-AgNCs decreased slightly. Therefore, the dual emission fluorescence sensing system was constructed based on a fluorescence substance and non fluorescence substance, to achieve determination of BER. Meanwhile, based on the bridging effect of BER and fluorescence resonance energy transfer effect from Dau, the altering of two peaks intensity was utilized to achieve determination of Dau. Thus, this dual emission sensing system can not only be used for fluorescence analysis of BER and its analogues, but also based on the bridging effect of BER, allowing the determination of Dau and its analogues that could not be directly measured with silver nanoclusters, expanding the application range of traditional dual emission detection systems. Meanwhile, this system has strong anti-interference ability and low toxicity to the human body and less pollution to the sample and environment. This provides a new direction and universal research strategy for the construction of new fluorescence sensing systems in the future for the analysis of target substances that cannot be directly detected with conventional fluorescence analysis methods.