【Backgrounds】 Bladder cancer (BC) is a kind of malignant tumor derived from the mucous membrane of the bladder. Moreover, it occurs frequently and is prone to recurrence. In especially, muscle-invasive BC progresses quickly and has a high risk of metastasis. However, there are few suitable experimental model cells. Organoid culture method has been applied to tumor research in recent years. In the previous study, we established a novel muscle-invasive BC organoid culture model using the urine samples from BC diseased dogs. Therefore, we focused on generating the urine sample-derived BC organoids from human BC patients 【Materials and methods】 To generate urine sample-derived human BC organoid, we collected the cells from bladder lavage fluid of human BC patients and tried to culture them. The composition of the culture medium was changed to more optimal components. 【Results and discussions】 The organoids created from bladder lavage fluid has no bacterial contamination and showed stable proliferation. We observed several bladder tissue marker expressions in the organoids and evaluated the difference in cell viability for the anti-cancer drugs. In conclusions, the organoids created from the bladder lavage fluid will be able to be taken advantage of as a valuable experiment model for human BC patients.