Today, a lot of attention is being paid to the pre-miRNAs/miRNAs or activity of Dicer due to their important functions in various physiological processes. Especially, the intrinsic relationship among these associated targets is of significant importance for more in-depth research on the mechanism of disease formation and early diagnosis. Herein, a strategy for simultaneous bioanalysis of miRNAs/pre-miRNAs and Dicer enzyme based on the self-designed multi-path nucleic acid amplification technology was proposed. Typically, in the presence of pre-miRNA-155, it can hybridize with Helper to generate a structure with two new toeholds, one of which could react with H1, H2, and H3, performing a modified CHA reaction with obvious fluorescence responses of FAM, and another of which could hybridize with H4, H5, and H6 to construct the [H4-H5-H6]n DNA nanosphere with obvious fluorescence responses of Cy5. Similarly, miRNA-155 could just hybridize with H1, H2, and H3 to generate the same modified CHA reaction with obvious fluorescence responses of FAM. Due to the successful multi-path nucleic acid amplification, the proposed bioanalysis strategy could be successfully employed for miRNA-155 and pre-miRNA-155 analysis in the range from 500 pM to 100 nM and 1 to 300 nM, respectively. The proposed strategy could be applied to explore another inter-related nucleic acid relationship also, providing great potential in bioanalysis of various nucleic acids.