For high-throughput screening of enzymes expressed intracellularly in E. coli, a facile, consistent, reagentless and in situ cell lysis method was developed by combining the autolytic gene cassette SRRz from bacteriophage lamda with a series of UV- and heat-inducible promoters to construct E. coli autolytic vectors. Previously constructed UV- and heat-inducible E. coli autolytic vectors led to lysis efficienc of 60% and 90%, but restricted cell growth to be 30°C or below. Aiming to broaden the application of this heat-inducible autolytic vector series, three additional autolytic vectors with heat-inducible promoters cI857/pR(T41C), htpG, and clpB, respectively were further tested. E. coli BL21 cells harboring these vectors grew well at 35°C and lysed efficiently (94%-98%) within 1-2 h after heat induction for 30 min at 42°C or 45°C. Promoter cI857/pR(T41C) performed best with the highest lysis efficiency and lowest basal level lysis, while htpG showed a slightly more background lysis, and clpB preferred a higher heat induction temperature of 45°C instead of 42°C. Application of the autolytic vector using cI857/pR(T41C) as promoter either in 96-well plates, or on nitrocellulose membranes, or on agar plates led to facile, efficient and consistent release of intracellular recombinant enzymes (e.g., lysis efficiency of 91.8% ± 1.1% in 96-well plates). Further application in directed evolution was illustrated by improving the thermostability of amadoriase using this vector. This reagentless and in situ cell lysis method also has the potentials for lysis of miniaturized samples in clinical diagnosis and bioanalytical detection, and for lysis of cells in the microarray format.