The invention provides a nitrogen, phosphorus and sulphur doping or co-doping carbon dot and a batch controllable preparing method and application thereof. The method comprises the steps that a carbon source, a nitrogen source, a phosphorus source and a sulphur source are evenly mixed, and a mixture is obtained, wherein the molar ratio of C to N to P to S in the mixture is 1 to 0-0.8 to 0-0.4 to 0-0.4, and the contents of N, P and S are prevented from being zero at the same time; in the air, the mixture is heated to be fused, the reaction is carried out for 3 min to 60 min, natural cooling is carried out till the indoor temperature is reached, a reaction product is separated by a silicagel column, raw materials which do not react are removed, and the nitrogen, phosphorus and sulphur doping or co-doping carbon dot is obtained. According to the method, the technology is simple, the compound time is short, batch producing can be achieved, the doping amount can be adjusted and controlled accurately, the fluorescence color of the prepared carbon dot ranges from blue to green, the application can be achieved on bioluminescence marking and cell imaging aspects, and the good economic benefit and the application prospect are achieved.