Biomembranes constitute a basis for all compartments of live cells, and therefore monitoring their lipid organization is essential for understanding cell status and activity. However, sensing and imaging lipid organization specifically in different organelles of live cells remains challenging. Here, we designed an array of solvatochromic probes based on Nile Red bearing ligands for specific targeting of endoplasmic reticulum, mitochondria, lysosomes, Golgi apparatus, plasma membranes and lipid droplets. These polaritysensitive probes detected variations in the lipid order by changing their emission maximum, as evidenced by fluorescence spectroscopy in model membranes. In colocalization microscopy experiments with reference organelle markers, they exhibited good organelle selectivity. Using two-color fluorescence microscopy, the new probes enabled imaging local polarity of organelles in live cells. To exclude the biased effect of the probe design on the sensitivity to membrane properties, we calibrated all probes in model membranes under the microscope, which enabled first quantitative description of lipid order in each organelle of interest. Cholesterol extraction/enrichment confirmed capacity of the probes to sense lipid order, revealing that organelles poor in cholesterol are particularly affected by its enrichment. The probes also revealed that oxidative and mechanical stresses produced changes in the local polarity and lipid order that were characteristic for each organelle, with mitochondria and lysosomes being particularly stresssensitive. The new probes constitute a powerful toolbox for monitoring the response of the cells to physical and chemical stimuli at the level of membranes of individual organelles, which remains an underexplored direction in the cellular research.