Harmful algal blooms caused by Karlodinium veneficum recently occurred with high incidence, posing a serious threat to the marine ecological environment, public health, and mariculture. It is therefore rather vital to establish a method for rapid detection of K. veneficum. In this study, the D1-D2 region of the large subunit rDNA (LSU rDNA D1-D2) of K. veneficum was cloned and sequenced to design the specific probes and primers. A novel method referred to as double-nick rolling circle amplification (dn-RCA) based on the designed probes and primers was initially established. The optimal reaction conditions for dn-RCA were as follows: probe concentration, 200 pM; ligation temperature, 57 °C; ligation time, 50 min; amplification temperature, 60 °C; and amplification time, 60 min. Furthermore, lateral flow dipstick (LFD) was employed instead of agarose gel electrophoresis to analyze dn-RCA products, which can simplify the detection procedure and reduce the operation time. The sensitivity of dn-RCA-LFD was tested with the genomic DNA, the recombinant plasmid containing the inserted LSU rDNA D1-D2, and the DNA crude extract of K. veneficum. The results showed that the sensitivity of dn-RCA-LFD was 10 times higher than that of conventional PCR; the detection limit of dn-RCA-LFD was 1.1 × 10-4 ng µL-1 for the genomic DNA, 360 copies µL-1 for the recombinant plasmid, and 5.3 cells mL-1 for DNA crude extract. The results of the cross-reactivity test with 22 control microalgal species showed that the dn-RCA-LFD had high specificity for K. veneficum. The stability of dn-RCA-LFD was tested by mixing the interfering genomic DNA with the target genomic DNA, which can be expected to simulate the natural samples containing different ratios of interfering cells to target cells. The results indicated that the performance of dn-RCA-LFD was immune to the DNA concentration of the interfering species. Finally, the practicability of dn-RCA-LFD was further confirmed by the test with field samples collected from the East China Sea. In conclusion, the established dn-RCA-LFD has advantages of high sensitivity, strong specificity, and stable performance, and is therefore promising for rapid detection of K. veneficum.