Purpose The analysis covered the interaction of humic acids (HA) isolated from peats with the metazachlor using spectroscopic methods. Materials and methods Five peatlands have been selected for the study in the southern and northern parts of Poland. Extraction of humic acids (HA1, HA2, HA3, HA4, and HA5) was performed using a procedure recommended by the International Humic Substances Society. Metazachlor 500 SC with metazachlor as a biologically active substance [2-chloro-N-(pyrazol-1-ylmethyl)acet-2′,6′-xylidide] has been used. HA have been assessed on the basis of their elemental composition. Spectroscopic techniques have been used to study the HA structure and their interaction with metazachlor including: UVvisible spectroscopy, dynamic light scattering, and delayed luminescence (DL) Results and discussion Based on their morphological and chemical properties, the studied peat soils can be classified as follows: (1) Eutric Murshic Sapric Histosols, (2) Eutric Sapric Histosols, (3) Eutric Hemic Histosols, (4) Eutric Fibric Histosols, and (5) Dystric Dranic Hemic Histosols according to the WRB guidelines (2015). Results showed that the largest particle size, measured by E 4/6 coefficient, was found in HA2 sample, while the smallest in HA3 sample. The addition of metazachlor to HA solutions reduced the values of E 4/6 coefficient by 33 to 72%, while the values of hydrodynamic diameter by 4 to 32%. No significant changes were found between E 4/6 after adding of metazachlor to HA solutions. The largest changes in DL intensity, excited both by blue and red light after herbicide addition, were observed in HA2 sample, which is characterized by the largest particle size, the highest oxygen content and CQ value as well as the lowest Baromatization rate^value. Conclusions The particle sizes of HA molecules measured by E 4/6 coefficient do not impact on the particle sizes of HA-herbicide systems measured by the same ratio. The delayed luminescence may become valuable, because it is simple, fast, and sensitive. Additionally, the results show minor errors comparing with chemical methods. This method may provide information about structure and nature of humic acids, as well as their photoreactivity. The using of delayed luminescence in studies of HApesticides interaction may enable us to better understand the luminescence properties of HA.