Abstract Introduction/Objective Pesticides use has been an integral part of modern agricultural practices. Lack of knowledge and unsafe handling of pesticides uses have kept the farm workers in the frontline of vulnerability. Serum cholinesterase show a dose-exposure relationship with organophosphorus. This study aims to identify the existing knowledge, attitude and practices of pesticides use among farm workers and to measure the serum cholinesterase level before and after spraying of the pesticides. Methods/Case Report This is a cross-sectional study carried out among farm workers of Madhyapur Municipality, Bhaktapur, Nepal. Ethical clearance was obtained from Nepal Health Research Council, Nepal. House hold survey was carried out for the knowledge, attitude and practices of pesticides use using structured questionnaires. Written consent was taken. Blood was collected twice, before and after spraying season for 97 farm workers. Serum cholinesterase was analyzed in the vitros 4600 biochemistry analyzer. Paired t-tests and chi square tests were used for statistical analysis. Results (if a Case Study enter NA) The mean cholinesterase of farm workers before and after pesticide spraying was 6.92±1.47 U/mL and 6.49±1.47 U/mL respectively, with p value of <0.001. There were also significant differences of serum cholinesterase level among farm workers before and after pesticide spraying on the basis of education level, type of involvement either full time or part time and prior integrated pest management training. This study found that 63% of respondents had good knowledge on pesticides while 25% had average and 12% had poor knowledge. Similarly, this study also found that 54% of respondents had good knowledge on pesticides use while 26% had average and 20% had poor knowledge. However, this study showed that only 44% of farm workers had good practice. Association between knowledge of pesticide and practices and between knowledge of pesticides use and practices were statistically significant with p-value of 0.001 and 0.001 respectively. Conclusion There is a significant difference in the level of serum cholinesterase before and after spraying pesticides in farm workers. Lack of education, lack of integrated pest management training and poor practices of pesticides use are the reasons behind the difference in the serum cholinesterase level.