The invention discloses a cis tetraphenylethylene macrocyclic bisquaternary ammonium salt and preparation and application methods thereof and belongs to the technical field of biochemical detection. The preparation method comprises heating cis dichloromethylene tetraphenylethylene crown ether or cis dibromomethylene tetraphenylethylene crown ether and triethylamine at 70-100 DEG C for 4-20 h for nucleophilic substitution, removing solvent through evaporation and then performing recrystallization to obtain the cis tetraphenylethylene macrocyclic bisquaternary ammonium salt. The cis tetraphenylethylene macrocyclic bisquaternary ammonium salt is in a cis cyclic structure, so that when two quaternary ammonium salt groups are aggregated onto DNA (deoxyribonucleic acid) chains through electrostatic interaction, not only is rotation of unit benzene rings of tetraphenylethylene restricted, but also rotation of the double bond of the tetraphenylethylene can be hampered, further the cis tetraphenylethylene macrocyclic bisquaternary ammonium salt can be ultrahigh in sensitivity when applied to detecting DNA; when the DNA contains 30 basic groups, the minimum concentration of the cis tetraphenylethylene macrocyclic bisquaternary ammonium salt for DNA detection is 32 pmol/L.