The invention discloses attenuated, brightened and replication-controllable HSV recombinant virus, a preparation method and applications thereof. According to the present invention, thymidine kinase (TK) gene essential for replicating viruses in neurons and being a main virulence factor is knocked out by using a homologous recombination method, and subsequently a red or green fluorescent gene enhancement expression cassette is recombined into the genome of the virus to construct a series of novel recombinants viruses, wherein the toxicity is markedly low, the states of infected mice are good,the fluorescence signal is strong, and the expression of the recombinant viruses is limited at the injection site after the recombinant viruses are used in in-vivo animal center labeling; by combiningwith Cre-dependent AAV helper viruses capable of expressing TK in a compensated manner, the cell-specific transmonosynaptic loop tracing is achieved; and the recombinant HSV has wide application value in nervous system targeted gene transduction, neural network transsynaptic tracing, tumor disintegration, viral replication and pathogenesis mechanism, antiviral drug screening and other fields.