CRISPR/Cas systems have displayed remarkable potential in developing novel biosensing applications for nucleic acid detection owing to the collateral cleavage activity of Cas effector proteins (Cas12, Cas13, etc.). Despite tremendous progress in recent years, the existing CRISPR/Cas based biosensing platforms have several limitations, including reliance on proper amplification methods, expensive fluorescence detection equipment, or lateral flow biosensor (LFB). Herein, we report a simple, inexpensive, and ultrasensitive DNA probe based LFB with CRISPR/Cas and loop-mediated Isothermal Amplification (namely CIA). The concept behind this approach is a non-detectable test line on the LFB when the Cas effector protein collaterally cleaves the cognate target and an ssDNA reporter sequence. The CIA based LFB can detect as low as a single copy cloned Pseudomonas aeruginosa acyltransferase gene, 1 cfu/ml plasmid containing E. coli DH5 alpha pure cultures, as well as clinical samples without DNA extraction/purification or advanced apparatuses. No cross-reactivity with other non-target bacteria was observed. The naked eye result readout was obtained in 15 min of LAMP amplification, 30 min of Cas12 reaction, and 5 min of LFB readout. This platform is robust and of low cost for on-site testing.