Harmful algal blooms (HAB) involving toxic microalgae have posed a serious threat to the marine industry and environment in the past several decades. Efficient techniques are required to monitor the marine environment to provide an effective warning of imminent HAB. Sequenced the partial large subunit rDNA (D1-D2) sequences of eight toxic harmful algae that are commonly distributed along the Chinese coast were cloned. Specific padlock probes (PLP) that contain linker regions composed of universal primer binding sites and Zip sequences were designed from the obtained target DNA. Taxonomic probes complementary to the Zip sequences were tailed and spotted onto a nylon membrane to prepare a DNA array. An optimized multiplex hyperbranched rolling circle amplification (MHRCA) was used to produce biotin-labeled amplified products. Heat-denatured MHRCA products were used to hybridize with DNA array, followed by dot coloration. An MHRCA-based membrane DNA array assay (MHBMDAA) for detecting toxic microalgae was developed. The specificity of the MHBMDAA was confirmed by double cross-reactivity tests of PLP and taxonomic probes. The MHBMDAA was competent for detecting the simulated samples with 10³ to 10⁻¹ cells mL⁻¹, which is 10-fold more sensitive than a multiplex PCR-based membrane DNA array. The effectiveness of the MHBMDAA was also validated by testing with natural samples from the East China Sea. Results indicated that the MHBMDAA provides a valuable tool for the sensitive and reliable detection of toxic microalgae for early warning and research purposes.