Rapid determination of chlorophyll content is significant for evaluating cotton's nutritional and physiological status. Hyperspectral technology equipped with multivariate analysis methods has been widely used for chlorophyll content detection. However, the model developed on one batch or variety cannot produce the same effect for another due to variations, such as samples and measurement conditions. Considering that it is costly to establish models for each batch or variety, the feasibility of using spectral preprocessing combined with deep transfer learning for model transfer was explored. Seven different spectral preprocessing methods were discussed, and a self-designed convolutional neural network (CNN) was developed to build models and conduct transfer tasks by fine-tuning. The approach combined first-derivative (FD) and standard normal variate transformation (SNV) was chosen as the best pretreatment. For the dataset of the target domain, fine-tuned CNN based on spectra processed by FD + SNV outperformed conventional partial least squares (PLS) and squares-support vector machine regression (SVR). Although the performance of fine-tuned CNN with a smaller dataset was slightly lower, it was still better than conventional models and achieved satisfactory results. Ensemble preprocessing combined with deep transfer learning could be an effective approach to estimate the chlorophyll content between different cotton varieties, offering a new possibility for evaluating the nutritional status of cotton in the field.