ObjectiveTo test the relation between adenosine triphosphate (ATP) biofluorescence detection technology and bacterial colony forming unit (CFU) and blood content.MethodsEscherichia coli （E. coli） ATCC  8099 and heathy controls’ blood were diluted to the concentration of 10-1,10-2,10-3,10-4,10-5,10-6 and 10-7, respectively, then lysate, luciferase and ATP standard substance were added, relative light unit (RLU) values were determined twice by fluorimeter, ATP contents(amol) were converted according to formula(ATP content=A1A2-A1×106); hemoglobin values were measured by hematology analyzer to reflect protein residue level. Curve demarcate standardization was made to show the relation between diluted bacteria, hemoglobin content and ATP content.ResultsBacterial CFU and ATP content logarithm values (y=1.07x-0.55,R2=0.99), bacterial CFU and ATP RLU logarithm values (y=1.14x+0.33,R2=0.99) showed linear relationship respectively. There was no significant difference between the logical values figured out by different RLU values and the actual values. After hemodilution, hemoglobin and ATP RLU logarithm values also showed linear relationship (y=1.03x-8.42, R2=0.99).ConclusionATP biofluorescence detection technology can detect the content of bacteria and protein through ATP value and RLU, it can determine contamination degree and clean effect of medical equipments and object surface, it’s a new， sensitive and rapid detection method.