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An electrochemical biosensor was fabricated for reliable and highly sensitive microRNA-21 assay by catalytic hairpin assembly and Pd nanoparticles-DNA layered nanoreticulation, providing a new way in the biochemical assay for detecting microRNA-21 in early disease diagnosis. • The sensitive biosensor was constructed for the detection of miR-21 based on CHA and Pd NPs-DNA LNR. • This biosensor shows low limit of detection and wide dynamic correlation of miR-21, providing a powerful platform for detecting miR-21. • The proposed method was enzyme-free, PCR-free, and convenient without the requirement of any additional separation steps. Herein, a Pd nanoparticles-DNA layered nanoreticulation (Pd NPs-DNA LNR) sensor was assembled to a graphene oxide (GO)-modified electrode by catalytic hairpin assembly (CHA) and a self-assembly process. In this process, GO was modified on the electrode surface to provide -COOH sites for DNA bonding. Moreover, with the CHA circular amplification strategy, many single DNA1 were exposed to be able to be bound with Pd-DNA2 for the formation of the Pd NPs-DNA LNR, which enabled the ultrasensitive detection of microRNA-21 (miR-21) with a low limit of detection 63.1 aM. This strategy combines the amplification technology of CHA and Pd NPs-DNA LNR with Pd NPs. Such a layered nanoreticulation material can support abundant Pd NPs to avoid aggregation and obtain enhanced electrochemical signals. Significantly, this method can be successfully applied in tumor cell lysates and provides a neoteric channel for early disease diagnosis. [ABSTRACT FROM AUTHOR]