Rapid and accurate detection of the causes of gastrointestinal diseases in farmed and companion animals is crucial for advancing livestock farming and safeguarding public health safety. Diseases caused by pathogenic bacteria infections often result in the overrepresentation of pathogens in the gut microbiota; however, gut microbiota dysbiosis without obvious pathogen overrepresentation can also lead to disorders such as inflammatory bowel disease (IBD). Traditional cultivation-based diagnostic methods are time-consuming and ineffective in identifying microbiota dysbiosis-associated diseases. In this study, we developed a sample-to-answer MinION full-length 16S rDNA sequencing analysis pipeline, accompanied by detailed bioinformatics scripts, for the rapid diagnosis of animal gastrointestinal diseases. The pipeline enables the detection of pathogens and microbiota dysbiosis-associated diseases in approximately six hours. The pipeline showed high sensitivity and specificity, as evident by the analysis of artificially contaminated samples, and accurately diagnosed bacterial infections in five cases, including chicken, duckling, and piglet samples from their respective farms, as well as a companion cat, outperforming traditional methods. It also rapidly identified IBD in five companion animals. The findings highlight the potential application of our developed sample-to-answer analysis pipeline in pathogen detection and the diagnosis of gut microbiota dysbiosis-related diseases in animals, thereby improving livestock health and public safety.