The objective of this study was to examine the structure changes in humic acids (HAs) in Chernozem after the application of different types of organic materials (OMs) under an indoor simulation condition for plastic mulched drip irrigation, measured with Fourier transform infrared (FTIR) spectroscopy. The biotechnological extract of fulvic acid (BFA), decomposed sheep manure (M), corn straw pellets (Ps) and corn straw powder (Pr) were used as the four OMs for testing, and they were applied to Chernozem at the same amount of actual material; three nitrogen (N) levels (no N, low N, and high N supply) were applied to each type of (OMs), separately. The total culture period was set to 90 days and soil sampling was taken at 0, 30, 60 and 90 days, respectively. The results showed that different types of OMs exerted different effects on Chernozem based on the FTIR spectra of HAs. The application of M combined with high N supply was the best way to fertilize Chernozem, under which the H-bonded OH groups and aromatic compounds were enhanced, resulting in increased soil carbon (C) sequestration; while the carbohydrates in HAs was easily consumed as microbial energy substance. The HAs from the Chernozem amended with BFA became more aliphatic, simpler and younger. High N supply was beneficial for increasing the complexity of HAs from Chernozem amended with Ps, but was not conducive to soil cation retention. Within a short time of incubation, the application of Pr combined with high N was detrimental to the C sequestration in Chernozem, and inhibited the consumption of carbohydrates by microorganisms.