Non-ionic surface active agents are a diverse group of chemicals which have an uncharged polar head and a non-polar tail. They have different properties due to amphiphilic structure of their molecules. Commercial available non-ionic surfactants consist of the broadest spectrum of compounds in comparison with other types of such agents. Typically, non-ionic compounds found applications in households and industry during formulation of cleaning products, cosmetics, paints, preservative coatings, resins, textiles, pulp and paper, petroleum products or pesticides. Their are one of the most common use class of surfactants which can be potential pollution sources of the different compartment of environment (because of they widely application or discharging treated wastewaters to surface water and sludge in agricultural). It is important to investigate the behavior, environmental fate of non-ionic surfactants and their impact on living organisms (they are toxic and/or can disrupt endocrine functions). To solve such problems should be applied appropriated analytical tools. Sample preparation step is one of the most critical part of analytical procedures in determination of different compounds in environmental matrices. Traditional extraction techniques (LLE -for liquid samples; SLE -for solid samples) are time and solvent-consuming. Developments in this field result in improving isolation efficiency and decreasing solvent consumption (eg SPE and SPME -liquid samples or PLE, SFE and MAE -solid samples). At final determination step can be applied spectrophotometric technique, potentiometric titrametration or tensammetry (determination total concentration of non-ionic surfactants) or chromatographic techniques coupled with appropriated detection techniques (individual analytes). The literature data concerning the concentrations of non-ionic surfactants in the different compartments of the environment can give general view that various ecosystems are polluted by those compounds.